NZ786946A - Novel peptides and combination of peptides for use in immunotherapy against aml - Google Patents

Abstract

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Description

Novel es and combination of peptides for use in immunotherapy against AML and other cancers This application is a onal of New Zealand patent application number 746667, the entire content of which is incorporated herein by this cross-reference.

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the t invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumorassociated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

The present invention relates to several novel peptide sequences and their variants d from HLA class I les of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses, or as targets for the development of pharmaceutically / immunologically active compounds and cells.

BACKGROUND OF THE INVENTION Acute myelogenous leukemia/ acute myeloid leukemia (AML) is a myeloproliferative disease that is characterized by an elevated count of immature myeloid blasts in bone marrow and peripheral blood. It is the most common type of leukemia in adults and accounts for imately 25% of all leukemias in the Western World. The incidence is t in the US, Australia and Western Europe, with about 3.8 cases per 100,000 in the US and Europe (Deschler and t, 2006; Showel and Levis, 2014).

AML is a disease of the elderly: The incidence for adults over 60 years is 15 cases per 0 (Showel and Levis, 2014). Patients newly diagnosed with AML have a median age of 67 years (American Cancer Society, 2015). Males have a slightly higher risk for developing AML in most countries ler and Lubbert, 2006).

AML has the lowest survival rate of all leukemias ler and Lubbert, 2006). The 5- year overall survival (08) in patients older than 75 is less than 10% with no improvement over the last 30 years. The 5-year OS for patients aged 25 to 39 has ed from less than 10% to 50% (Showel and Levis, 2014). The ity rate for males is higher than for females. AML mortality is greater in whites than in blacks (Deschler and Lubbert, 2006).

AML is diagnosed when at least 20% (World Health Organization (WHO) classification) or at least 30% (French-American-British (FAB) classification) blasts of the myeloid lineage are present in bone marrow or blood. Diagnosis is done on blood or bone marrow samples. Tests include a complete blood count and copic exams, cytochemistry, immunohistochemistry, flow cytometry, reverse riptase polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH) (National Cancer Institute, 2015).

Symptoms for AML are often unspecific and include weight loss, loss of appetite, fatigue, fever, hes and sleepiness. Risk factors for developing AML include smoking, male gender, exposure to benzene, chemotherapeutical ent with alkylating agents or topoisomerase II inhibitors and radiation exposure (National Cancer Institute, 2015).

As AML is a very heterogenous disease there is no unique classification system. The WHO classifies AML according to morphology, cytogenetics, molecular genetics and immunologic markers. The FAB system fies AML using morphology as determined by the degree of differentiation and the extend of cell maturation. Depending on the above-named criteria the following AML subtypes exist according to WHO and FAB (National Cancer Institute, 2015): Percentage of Classification Description AML with t(8;21)(q22;q22) (AML/ETO) 5-12% AML with AML with )(p13q22) or t(16;16)(p13;q22) -12% characteristic (CBF /MYH11) genetic APL: AML with t(15;17)(q22;q12) (PML/RARA and alities variants) AML with 11q23 (MLL) abnormalities 5-6% AML with AML with FLT3 mutations (FLT3/ITD: 23%, FLT3 -30% genetic point mutation: 7%) abnormalities AML with NPM1 mutations in FLT3, AML with CEBPA mutations NPIVH, AML with t(9;11)(p22;q23) (MLLT3/MLL) CEBPA, AML with t(6;9)(p23;q34) (DEK/NUP214) MLLT3/MLL, AML with (q21q26.2) or t(3;3)(q21;q26.2) DEK/NUP214, EVI1) RPN1/EVI1, AML with t(1;22)(p13;q13) (RBM15/MKL1) AML evolving from MDS or following MDS, variants: AML with complex karyotype, AML with deletions/ aberrations of/in the following chromosomes: _ del(7q), del(5q), i(17q)7t(17p), del(13q), del(11q), AML With p)/t(12p), del(9q), idic(X)(q13), AML wuth. multilineage_ _ translocations between the follownng chromosomes:, dysplasia t(11;16)(q23;q13.3), t(3;21)(q26.2;q22.1), t(1;3)(p36.3;q21.1), t(2;11)(p21;q23), t(5;12)(q33;p12), t(5;7)(q33;q11-2), t(5;17)(q33;p13), t(5;10)(q33;q21), t(3;5)(q25;q34) AML and Alkylating agent-related AML and MDS MDS, therapy Topoisomerase ll inhibitor-related AML _ 4 _ related Acute myeloblastic leukemia, minimally differentiated Acute lastic leukemia, without maturation 10% Acute myeloblastic leukemia, with maturation 30-45% Acute myelomonocytic leukemia (AMML) 15-25% AML not Acute monoblastic leukemia and acute monocytic ise leukemia categorized Acute oid leukemias 5-6% Acute megakaryoblastic leukemia, variant: transient myeloproliferative disorder in Down syndrome Acute basophilic leukemia <1% Acute panmyelosis with myelofibrosis Myeloid sarcoma 2—8% Acute Acute leukemia of rmined lineage leukemias of Acute leukemia with mixed phenotype ambiguous Acute leukemia with mixed lineage lineage Hybrid acute leukemia Classification Name Cytogenetics acute myeloblastic leukemia, minimally differentiated M1 acute myeloblastic leukemia, without maturation M2 acute myeloblastic leukemia, With tion. . . . t(8;21)(q22;q22), t(6'9) M3 acute promyelocytic leukemia (APL) t(15;17) _ _ )(p13q22), M4 acute myelomonocytic leukemia (AMML) del(16q) M4(Eo) acute myelomonocytic leukemia with bone marrow inv(16), t(16;16) _ 5 _ eosinophilia M5a and acute monoblastic leukemia and acute tic del(11q), t(9;11), M5b leukemia t(11;19) M6a and acute erythroid leukemias M7 acute megakaryoblastic leukemia t(1;22) M8 acute basophilic ia AML treatment is divided into two phases: induction therapy and post-remission/ "consolidation therapy". lnduction therapy is administered to induce remission and consists of combinational chemotherapy. Consolidation therapy consists of onal chemotherapy or hematopoietic cell transplantation (HCT) l and Levis, 2014).

The most common chemotherapeutic drugs used to treat AML are cytarabine, daunorubicin, idarubicin and mitoxantrone followed by cladribine, fludarabine and diverse others. Azacitidine and decitabine (DNA hypomethylating agents) are now used for treatment of MDS/AML. Treatment for APL/ AML M3 es all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) (National Cancer ute, 2015).

"Standard treatment" for AML is considered as "3+7": 3 days of idarubicin or daunorubicin and 7 days of cytarabine, followed by l similar courses to achieve complete remission (CR) , 2014). The decision between standard y and al trial is based on the risk stratification. The European Leukemia Net (ELN) system distinguishes between the following prognostic groups: Prognistic goup Subsets inv(16) or t(16;16); t(8;21); NK and NPM1+/FLT3 lTD-; NK and "Favorable" CEBPA+/+ Intermediate-1 NK and NPM1-/FLT3 |TD-; CEBPA+/- Cytogenetic abnormalities not in "favorable" or "adverse" groups; Intermediate-2 FLT3 lTD+ -5, -7, 5q-, abn 3q, 17p, 11q (other than 9;11), , complex; "Adverse" insufficient metaphases for analysis AML cases with intermediate-risk karyotype show either no ypic abnormalities or only one or two abnormalities not categorized as high- or low-risk.

FLT3 mutations are associated with an aggressive type of AML and a poor prognosis.

They often occur together with NPM1 and DNMT3a (DNA methyltransferase 3A) mutations. NPM1 (nucleophosmin) mutations are a favorable prognostic indicator, if not found together with FLT3 mutations. CEPBA (CCAAT-enhancer—binding protein alpha/ C/EBPd) ons confer a survival age in the case of double or homozygous CEBPA mutations without wild-type expression. Altered methylation patterns in a variety of genes are caused by mutations in isocitrate dehydrogenase (IDH1 and lDH2) and DNMT3A. These mutations are associated with poor survival.

AML cases with favorable-risk karyotype consist of APL (acute promyelocytic leukemia) and CBF (core-binding factor) leukemias. APL cases are associated with the fusion of the myeloid transcription factor PML to the retinoic acid or subunit alpha .

The PML/RARA translocation is a favorable prognostic mutation. CBF leukemia cases show translocations involving a subunit of CBF. ln t(8;21) CBF alpha is fused to the ETC gene. In inv(16) CBF beta is fused to the smooth muscle myosin heavy chain. CBF ocations are very favorable prognostic mutations.

AML cases with unfavorable-risk karyotype are characterized by a complex karyotype including chromosomal aberrations such as ocations, nced rearrangements and gains/losses of whole somes. They are associated with a poor prognosis.

MDS/AML cases evolve from myelodysplastic syndromes and carry a worse prognosis than other AML sub-groups (Showel and Levis, 2014).

Besides the above-listed prognostic factors, additional molecular markers or marker combinations can be used to judge the prognosis in specific cytogenetic s: TP53 mutations are the most unfavorable genetic alteration in AML. NPM1 mutated and FLT3 WT together with a mutation in IDH1 or |DH2 is seen as favorable. rable factors include a partial tandem duplication in the MLL gene, a mutated TET2 gene, FLT3 lTD + together with a mutation in DNMT3a and CEBPA, FLT3 lTD — together with a mutation in ASXL1 or PHF6, and CD25 expression (stem cell-like "signature" and poorer outcome). The presence of CKIT mutations converts the prognosis of ts with a favorable inv(16) or t(8;21) into a more intermediate range. SPARC is up- regulated in NK (normal karyotype) patients with rable gene expression signature and down-regulated in association with the favorable NPM1 mutation. 5 over- expression s a poor prognosis in NK AML. Differentially methylated regions (DMRs) are prognostic when found in association with several genes (FLT3 mutation, NPM1 mutation). In this case, a lower expression is associated with a better sis (Estey, 2014).

Post-treatment information/ information about minimal al e (MRD) should be included into following treatment decisions. These include the response to induction therapy, PCR of fusion transcripts, mutated genes and over-expressed genes to detect MRD and multi-parameter flow try for observation of aberrant expression of surface antigens.

The following table combines AML prognostic groups and treatment recommendations: Prognostic group Subsets Induction Post-remission "Favorable" inv(16) or t(16;16); 3+7; consider ara-C (6 doses x 2 t(8;21); NK and FLAG-ida if age < courses), possibly NPM1+/FLT3 lTD-; 60-65 preceded by 1 NK and CEBPA+/+ course FLAG-ida Intermediate-1/lnt-1 NK and NPM1- 3+7; consider (a) HOT from matched /FLT3 lTD-; da if age < sibling donor (MSD) CEBPA+/— 60-65 or (b) clinical if risk NRM <20- trial 25%; if not HOT candidate FLAG-ida then ara-C or clinical trial Intermediate-2/ Int-2 Cytogenetic 3+7; consider (a) HCT from MSD or abnormalities not in FLAG-ida if age < URD if risk NRM < "favorable" or 60-65 or (b) clinical 30%; otherwise as "adverse" groups; trial; clinical trial intermediate-1; if FLT3 lTD+ combining FLT3+/ FLT3 lTD+ chemotherapy with consider FLT3 FLT3 inhibtor (e.g. inhibitor post HCT quizartinib, crenolanib) "Adverse" -5, -7, 5q-, abn 3q, Clinical trial HCT from MSD or 17p, 11q (other than URD if risk NRM < 9;11), t(6;9), 40%; consider post- complex; icient HCT trial; if not ases for HCT: candidate analysis clinical trial NRM: non-relapse mortality after HCT ent options for the prognostic groups favorable, intermediate-1 and possibly intermediate-2 include the "standard therapy 3+7", a combination of daunorubicin and cytarabine or idarubicin and cytarabine, or the administration of FLAG-ida, a ation of fludarabine, cytarabine, G-CSF and idarubicin. A third option is gemtuzumab ozogamicin (G0), which is a conjugate of an anti-CD33 monoclonal antibody and the cytotoxic agent calechiamicin. As CD33 is expressed in > 90% of ts with AML, the use of GO in combination with 3+7 or da may lead to longer ions and survival. Dasatinib is given in combination with 3+7 and then in _ g _ combination with high-dose cytarabine in patients with CKIT mutations in ) and t(8;21) AML (Estey, 2014).

Clinical trials are recommended for patients who belong to the prognostic groups unfavorable and intermediate-2. ent s include hypomethylating agents (HMAs) as azacitidine or decitabine, CPX-351, which is a liposomal formulation of daunorubicin and cytarabine in a 1:5 "optimal" molar ratio, and volasertib, which is an inhibitor of polo kinases. Volasertib is given in combination with LDAC (low-dose cytarabine). l different FLT3 inhibitors can be administered in case of FLT3 mutations. These include sorafenib, which is given in combination with 3+7, quizartinib, a more selective inhibitor of FLT3 lTD that also inhibits CKIT, anib, and midostaurin, an unselective FLT3 lTD inhibitor. r ent option is targeting CD33 with antibody-drug conjugates (anti-CD33 + calechiamicin, SGN-CD33a, anti- CD33 + actinium-225), bispecific antibodies nition of CD33 + CD3 (AMG 330) or CD33 + CD16) and chimeric antigen receptors (CARs) (Estey, 2014).

If possible, AML patients can undergo allogeneic hematopoietic cell transplantation (HCT). HCT in CR (complete ion) 1 should be performed if the expected reduction in risk of e is greater than the risk of HCT-related complications. HCT can also be performed in CR2 or with active disease, as some patients who relapse after not receiving HCT in CR1 can still be cured. Patients that are seen unfit for myeloablative (MA) intensity HCT (patients > 70-75, frail younger patients) can undergo a d intensity HCT (RIC-HCT). Possible donors are MUDs (matched unrelated donors), haploidentical donors or cells from umbilical cords that exceed the number of MSD (matched sibling donors). One third of patients have a HLA-matched sibling and about 50% have a potential matched unrelated donor (MUD) (Estey, 2014).

Considering the severe side-effects and expense associated with treating cancer, there is a need to identify factors that can be used in the ent of cancer in general and AML in particular. There is also a need to identify factors representing biomarkers for cancer in general and AML in particular, leading to better diagnosis of cancer, assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting of cancer cells while minimizing side effects. Cancer immunotherapy makes use of the existence of tumor associated antigens.

The t classification of tumor associated antigens (TAAs) comprises the following major groups: a) Cancer-testis antigens: The first TAAs ever identified that can be recognized by T cells belong to this class, which was originally called cancer-testis (CT) antigens because of the expression of its members in histologically different human tumors and, among normal tissues, only in spermatocytes/spermatogonia of testis and, occasionally, in ta. Since the cells of testis do not express class I and II HLA les, these antigens cannot be recognized by T cells in normal tissues and can therefore be considered as immunologically tumor-specific. Well-known examples for CT antigens are the MAGE family members and NY-ESO-1. b) Differentiation antigens: These TAAs are shared between tumors and the normal tissue from which the tumor arose. Most of the known differentiation antigens are found in melanomas and normal melanocytes. Many of these melanocyte lineage-related proteins are involved in thesis of melanin and are therefore not tumor specific but nevertheless are widely used for cancer immunotherapy. Examples include, but are not d to, tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer. c) Over-expressed TAAs: Genes encoding widely expressed TAAs have been ed in histologically different types of tumors as well as in many normal tissues, lly with lower expression . It is possible that many of the epitopes processed and potentially presented by normal tissues are below the threshold level for T-cell recognition, while their over-expression in tumor cells can trigger an anticancer response by breaking usly established tolerance. Prominent examples for this class of TAAs are Her-2/neu, survivin, telomerase, or WT1. d) Tumor-specific antigens: These unique TAAs arise from mutations of normal genes (such as B-catenin, CDK4, etc.). Some of these molecular s are associated with neoplastic transformation and/or progression. Tumor-specific antigens are generally able to induce strong immune responses without bearing the risk for autoimmune reactions against normal tissues. On the other hand, these TAAs are in most cases only relevant to the exact tumor on which they were identified and are usually not shared between many individual tumors. Tumor-specificity (or -association) of a peptide may also arise if the peptide originates from a tumor- (-associated) exon in case of proteins with tumor-specific (-associated) isoforms. e) TAAs arising from abnormal post-translational modifications: Such TAAs may arise from proteins which are neither specific nor overexpressed in tumors but nevertheless become tumor associated by posttranslational processes primarily active in tumors.

Examples for this class arise from altered glycosylation patterns g to novel epitopes in tumors as for MUC1 or events like protein ng during degradation which may or may not be tumor ic. f) Oncoviral proteins: These TAAs are viral proteins that may play a al role in the oncogenic s and, because they are foreign (not of human origin), they can evoke a T-cell response. Examples of such proteins are the human papilloma type 16 virus proteins, E6 and E7, which are sed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes d from tumor-associated or tumor-specific proteins, which are presented by molecules of the major ompatibility complex (MHC). The ns that are recognized by the tumor specific T lymphocytes, that is, the epitopes thereof, can be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc. which are expressed and, as compared to unaltered cells of the same origin, usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II. MHC class I molecules are composed of an alpha heavy chain and betamicroglobulin, MHC class II molecules of an alpha and a beta chain. Their three-dimensional conformation results in a binding groove, which is used for non-covalent interaction with peptides.

MHC class I les can be found on most nucleated cells. They present es that result from proteolytic cleavage of predominantly endogenous proteins, defective ribosomal ts ) and larger peptides. However, peptides derived from endosomal compartments or exogenous s are also frequently found on MHC class I les. This non-classical way of class I presentation is referred to as cross- presentation in the literature (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules can be found predominantly on professional antigen presenting cells (APCs), and ily present peptides of exogenous or transmembrane ns that are taken up by APCs e.g. during endocytosis, and are subsequently processed.

Complexes of e and MHC class I are recognized by CD8—positive T cells bearing the appropriate T-cell receptor (TCR), whereas complexes of peptide and MHC class II molecules are recognized by CD4-positive-helper—T cells bearing the appropriate TCR.

It is well known that the TCR, the peptide and the MHC are thereby present in a stoichiometric amount of 1:1 :1.

CD4-positive helper T cells play an important role in inducing and sustaining effective responses by CD8—positive cytotoxic T cells. The identification of CD4-positive T-cell epitopes derived from tumor associated antigens (TAA) is of great importance for the pment of pharmaceutical products for triggering anti-tumor immune responses (anatic et al., 2003). At the tumor site, T helper cells, support a cytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006) and attract effector cells, e.g. CTLs, natural killer (NK) cells, macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system, especially professional antigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells, macrophages, dendritic cells. In _ 13 _ cancer patients, cells of the tumor have been found to express MHC class II molecules (Dengjel et al., 2006).

Elongated (longer) peptides of the invention can act as MHC class II active epitopes.

T-helper cells, activated by MHC class II epitopes, play an important role in orchestrating the effector on of CTLs in anti-tumor immunity. er cell epitopes that r a T-helper cell response of the TH1 type support effector functions of CD8- positive killer T cells, which include cytotoxic functions directed t tumor cells displaying tumor-associated peptide/MHC complexes on their cell surfaces. In this way tumor-associated T-helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in the absence of CD8-positive T lymphocytes, CD4-positive T cells are sufficient for inhibiting manifestation of tumors via tion of angiogenesis by ion of interferon-gamma (lFNv) (Beatty and Paterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cells as direct anti-tumor effectors (Braumuller et al., 2013; Tran et al., 2014).

Since the constitutive expression of HLA class II molecules is usually limited to immune cells, the possibility of isolating class II peptides directly from primary tumors was usly not considered possible. However, Dengjel et al. were successful in identifying a number of MHC Class II epitopes directly from tumors ( EP 1 760 088 B1).

Since both types of response, CD8 and CD4 dependent, contribute jointly and istically to the anti-tumor effect, the identification and characterization of tumor- associated ns recognized by either CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or by CD4-positive T-helper cells (ligand: MHC class II le + peptide epitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immune response, it also must bind to an MHC-molecule. This process is dependent on the allele of the MHC-molecule and specific polymorphisms of the amino acid ce of the peptide. MHC-class-l- g es are y 8-12 amino acid residues in length and usually n two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC-molecule. In this way each MHC allele has a "binding motif" determining which peptides can bind specifically to the binding .

In the MHC class I dependent immune reaction, peptides not only have to be able to bind to n MHC class I molecules expressed by tumor cells, they uently also have to be recognized by T cells bearing specific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or -associated antigens, and to be used in a y, particular prerequisites must be fulfilled. The antigen should be expressed mainly by tumor cells and not, or in comparably small amounts, by normal healthy tissues. In a preferred embodiment, the peptide should be over-presented by tumor cells as compared to normal healthy tissues. It is furthermore desirable that the respective antigen is not only present in a type of tumor, but also in high concentrations (i.e. copy numbers of the respective peptide per cell). Tumor- specific and tumor-associated antigens are often derived from proteins directly involved in transformation of a normal cell to a tumor cell due to their function, e.g. in cell cycle control or ssion of apoptosis. Additionally, downstream targets of the proteins directly causative for a transformation may be up-regulated und thus may be indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets of a ation approach (Singh-Jasuja et al., 2004). It is essential that epitopes are present in the amino acid sequence of the antigen, in order to ensure that such a peptide ("immunogenic peptide"), being derived from a tumor associated n, leads to an in vitro or in vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as a T-cell epitope. A prerequisite for the ion of an in vitro or in vivo T-cell-response is the presence of a T cell having a corresponding TCR and the absence of immunological tolerance for this particular epitope.

Therefore, TAAs are a ng point for the development of a T cell based therapy including but not limited to tumor vaccines. The methods for identifying and characterizing the TAAs are usually based on the use of T-cells that can be isolated from patients or healthy ts, or they are based on the tion of differential ription profiles or differential peptide expression patterns between tumors and normal tissues. However, the identification of genes over-expressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not e precise information as to the use of the ns being transcribed from these genes in an immune therapy. This is because only an individual subpopulation of epitopes of these antigens are suitable for such an application since a T cell with a corresponding TCR has to be present and the immunological tolerance for this particular epitope needs to be absent or minimal. In a very preferred embodiment of the invention it is therefore important to select only those over— or selectively presented peptides against which a functional and/or a proliferating T cell can be found. Such a functional T cell is defined as a T cell, which upon stimulation with a specific antigen can be clonally ed and is able to execute effector functions ("effector T cell").

In case of ing peptide-MHC by specific TCRs (e.g. soluble TCRs) and antibodies or other g molecules (scaffolds) according to the invention, the immunogenicity of the underlying peptides is secondary. In these cases, the presentation is the ining factor.

SUMMARY OF THE INVENTION In a first aspect of the present invention, the present ion relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 188 or a variant sequence thereof which is at least 77%, preferably at least 88%, homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 188, n said variant binds to MHC and/or induces T cells cross-reacting with said peptide, or a pharmaceutical acceptable salt thereof, wherein said peptide is not the underlying full-length polypeptide.

The present invention further relates to a peptide of the present invention comprising a sequence that is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 188 or a variant thereof, which is at least 77%, preferably at least 88%, homologous (preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 188, wherein said peptide or variant thereof has an overall length of between 8 and 100, preferably between 8 and 30, and most red of between 8 and 14 amino acids.

The following tables show the peptides according to the present invention, their respective SEQ ID NOs, and the prospective source (underlying) genes for these es. All peptides in Table 1 and Table 2 bind to HLA-A*O2. The peptides in Table 2 have been disclosed before in large gs as results of high-throughput screenings with high error rates or calculated using algorithms, but have not been associated with cancer at all before. The peptides in Table 3 are additional es that may be useful in ation with the other peptides of the invention. The peptides in Table 4 are rmore useful in the diagnosis and/or treatment of s other malignancies that involve an over-expression or over-presentation of the respective underlying polypeptide.

Table 1: Peptides according to the present invention. J = phospho-serine SEQ ID No. Sequence GenelD(s) Official Gene Symbol(s) 1 YYL 2769 GNA15 2 VLLKAVAQA 3978 LIG1 3 ALYDKTKRIFL 1791 DNTT 4 FLPDAFVTM 1791 DNTT FLYYEDLVSC 1791 DNTT 6 GLAEVLLAA 4261 CIITA 7 LLWGDIMEL 79009 DDX50 8 LLWPGAALLV 7462 LAT2 _ 17 _ SEQ ID No. Sequence Gene|D(s) Official Gene Symbol(s) 9 SLLAYLEQA 57617 VPS18 VILDPVHSV 653659,92703 TMEM183B,TMEM183A 11 ILTQIDHIL 26574 AATF 12 ALIESNTAL 255631 1 13 ALVPGVTQV 64745 METTL17 14 ALWWGTITL 56479 KCNQ5 EDMYL 51244 CCDC174 16 FLDTQAPSL 116931 MED12L 17 FLLGLSEQL 389119 FAM212A 18 GIIEENWQL 221143 N6AMT2 19 GIVEYLSLV 5557 PR|M1 GLDAFLLEL 57674 RNF213 21 GLFHGTELL 5291 P|K3CB 22 GLLQLDTAFV 116115 ZN F526 23 GLLQPPVRIV 55749 CCAR1 24 GLVELLNRV 10636 RGS14 GVEGSLIVEKI 3329 HSPD1 26 SLIVEKI 3329 HSPD1 27 KANPALWL 10594 PRPF8 28 LLDQMETPL 1511 CTSG 29 GL 22984 PDCD11 SIISDSSAL 672 BRCA1 31 SLFVFIPMV 78992 YIPF2 32 SLSDRSWHL 29970 SCH|P1 33 TIMNQEKLAKL 23412652963 BTF3P16,BTF3P12,BTF3 34 TLSPWSFLI 79465 ULBP3 VLFEHAVGYAL 10528 NOP56 36 VLGPSPSSV 57597 BAHCC1 37 VVAPAPVVEAV 3609 ILF3 38 AAIASTPTL 79612 NAA16 39 AIFAGTMQL 55144 LRRC8D 40 ALAAGGYDVEKN 3009 HIST1 H1 B 41 ALFILPFVSV 10721 POLQ 42 ALTTYTIEV 54549 SDK2 43 AMLDFVSSL 55732 C1 orf1 12 44 FAVDNVGNRTL 55105 GPATCH2 45 FLFTDVLLM 84904 ARHGEF39 46 GLDQYLQEV 79825 CCDC48 47 GLIJPNVQL 79733 E2F8 48 IAIEALTQL 200424 TET3 WO 74645 SEQ ID No. Sequence Gene|D(s) Official Gene Symbol(s) 49 IIDDNHAIV ,5700 PSMC1 P4, PSMC1 50 IIWATSLLL 55728 N4BP2 51 SLLSSSLNV 55728 N4BP2 52 IVDPVDSTL 5591 PRKDC 53 KAFLGELTL 257218 SHPRH 54 KLPEFLVQL 203430 ZCCHC5 55 KTLDLINKL 57650 KIAA1524 56 LAN PTTSAL 8295 TRRAP 57 LLDFGSLSNLQV 53%520158503’4020 RPS17L,RPS17P16,RPS17 58 LLLATLQEA 790955 C11orf83 59 LSVPEGAIVSL 28672 TRAV12-3 60 NLLNVLEYL 167127 UGT3A2 61 FLLPGVLLSEA 167127 UGT3A2 62 RLLFNLSEV 253714 MMS22L 63 RLNDTIQLL 29128 UHRF1 64 SLANIKIWV 79895 ATP8B4 65 SLEEQLSALTL 8458 TTF2 66 SLKNEVGGLV 121278 TPH2 67 SLQDRVIAL 145508 CEP128 68 TGITTPVASV 23269 MGA 69 TIIGLVRVI 4998 ORC1 70 TLTDSNAQL 64105 CENPK 71 TLTSSLATV 79571 GCC1 72 VAFPSGDASSL 23312 DMXL2 73 VAI PDVDPL 777 CACNA1 E 74 VANPVLWL 11251 PTGDR2 75 VLAPLGFTL ,729148 NUS1 ,NUS1 P1 76 VLLJ PVPEL 64682 ANAPC1 77 VLNMKPPEI 10592 SMC2 78 VLSEVECHL 261734 NPHP4 79 YLMDPDTFTF 9582 3B 80 YLTEALQSI 5427 POLE2 81 YVTEELPQL 2098 ESD 82 LLPDNFIAA 2098 ESD 83 GLGAGVAEAV 6576 SLC25A1 84 GLLGSVLTI 8204 NR|P1 85 GLVPFGLYL 65250 C5orf42 86 HLLGDPMANV 10897 Y|F1A 87 NSI 3930 LBR 88 LALNFGSTL 201266 SLC39A11 SEQ ID No. Sequence Gene|D(s) al Gene Symbol(s) 89 LLESPVDGWQV 54892 NCAPG2 90 LLLDTVTSI 4130 MAP1A 91 RLAHYIDRV 84823 LMNB2 92 RLWDIQHQL 151790 WDR49 93 SLINDVLAE 340554 ZC3H12B 94 SLLEFAQYL 2175 FANCA 95 SVAEINVLI 221527 ZBTB12 96 TLLASYVFL 201305 SPNS3 97 TIMTGVIGV 201305 SPNS3 98 TQFGFLMEV 84674 CARD6 99 YLAPFSLSNY 79968 WDR76 100 GEVDTSLV 4353 MP0 101 AINKDPEAPIFQV 2108 ETFA 102 ALAQGAERV 23630 KCNE1 L 103 ALGDFGIRL 114548 NLRP3 104 ALIPETTTL 305292515119 CKLF-CMTM1,CKLF 105 GVFALVTAV 515119 CKLF-CMTM1,CKLF 106 ALLEELERSTL 9404 LPXN 107 ALLGMLPLL 8698 S1 PR4 108 ELEMNSDLKAQL 100128060,6201 RPS7P10,RPS7 109 GLLAVPLLAA 4232 MEST 110 GLTHTAWPLDLV 5250 SLC25A3 111 GVEPAADGKGVVVV 6158 RPL28 112 ILRDALNQA 6238 RRBP1 113 NLQSEVEGV 8673 VAMP8 114 RLAQEAAQV 114822 RHPN1 115 SLPDLTTPL 55904 MLL5 116 TILEILPEL 1462 VCAN 117 TILPTILFL 54497 HEATR5B 1 18 TLLTVLTQA 57508 |NTS2 119 AAL 51170 HSD17B11 120 VIQDLWSV 23556 PIGN 121 VLQAGQYGV 1786 DNMT1 122 VLL 9392 TGFBRAP1 123 YTVKINPTL 219972 MPEG1 124 GLPELVIQL 23279 NUP160 125 GLFGYLVFL 10312 TCIRG1 126 GLLPQQIQAV 25777 SUN2 127 KIISALPQL 152579 SCFD2 SEQ ID No. Sequence Gene|D(s) Official Gene Symbol(s) 128 NLSTKTEAV 140890 SREK1 129 EQV 4678 NASP 130 LEGV 4678 NASP 131 SLFSGSLEPV 6733 SRPK2 132 N FL 94005 PIGS 133 TVIGTLLFL 84876 ORAI1 134 AAGAGLPESV 9761 MLEC 135 GIIDRIFQA 6894 TARBP1 136 GLSSIETLL 1602 DACH1 137 ILAPLAWDL 11270 NRM 138 RQV 5987 TR|M27 139 NLIIFSPSV 1650 DDOST 140 YIPDFLTLL 64860 ARMCX5 141 GLLPPLRIPELL 79171 RBM42 142 GLSDGYGFTT 3185 HNRNPF 143 YLLPHILVY 545 ATR 144 GLFMGLVLV 56255 TMX4 145 VLLPLIFTI 146722 CD300LF 146 ALDTRVVEL 6320 CLEC11A 147 FALPILNAL 51202 DDX47 148 FLYFEDHGL 55255 WDR41 149 GLAEILVLV 9903 KLHL21 150 GLFGVLNEI 8086 AAAS 151 GLLPFPEVTL 25909,285116 AHCTF1,AHCTF1P1 152 GLSNHIAAL 29028 ATAD2 153 GLYTGQLAL 706 TSPO 154 IIADNIIFL 6742 SSBP1 015°"38' 783,1023 155 ILDLIQVFV 9,1176,348110 §§3S2,AP3S2,AP3S1,C15orf 156 ILMPELASL 5330 PLCB2 157 ILTETQQGL 26057 ANKRD17 158 LLLGGTALA 1991 ELANE 159 LLPLAPAAA 84519 ACRBP 160 RLVPFLVFV 3954 LETM1 161 SLIGIAIAL 9056 SLC7A7 162 SLLDFLTFA 23317 DNAJC13 163 SLMIDLIEV 57488 ESYT2 164 SLNPQEDVEF 84365 MKI67|P 165 SLVDRVAAA 4883 NPR3 166 VLFPLNLQL 1378,1379 CR1,CR1L WO 74645 _ 21 _ SEQ ID No. ce GenelD(s) Official Gene Symbol(s) 167 VLLDVALGL 9091 PIGQ 168 ALL 3326,3327 HSP90AB1 ,HSP90AB3P 169 VLQDPIWLL 5187,8864 PER1,PER2 Table 2: Additional peptides according to the present invention with no prior known cancer association. J = phospho-serine SEQ ID No. Sequence GenelD(s) Official Gene Symbol(s) 170 IVTEVAVGV 58155 PTBP2 171 DFL 55272 IMP3 172 KLLWGDIMEL 79009 DDX50 173 KMQETLVGL 8379 MAD1 L1 174 NLTENLQYV 55344 PLCXD1 175 KMDJFLDMQL 55728 N4BP2 176 HLWTGEEQL 146850 PIK3R6 177 QHV 64946 CENPH 178 KLWPLFVKL 55183 RIF1 179 ENL 641 BLM 180 ALDQEIIEV é13g045402525103364201280 2::1S71L7RPS17P16’RPS17P 181 KLLNHVTQL 79075 DSCC1 182 SLSEYDQCL 2769 GNA15 183 SVI GVSPAV 8567 MADD 184 RMTDQEAIQDL 4869 NPM1 185 RLI PI lVLL 64005 MYO1 G 186 llLDEAHNV 8458 TTF2 187 MLPPPPLTA 3978 LIG1 188 RLLDFPTLL 54627 KIAA1383 Table 3: Peptides useful for e.g. personalized cancer therapies.

SEQ ID No. Sequence GenelD(s) Official Gene Symbol(s) 189 RLFEEVLGV 9816 URB2 190 SLYKGLLSV 25788 RAD54B 191 ALSVLRLAL 6691 SPINK2 192 GLAALAVHL 2175 FANCA 193 FLLAEDTKV 10592 SMC2 194 LLWGNLPEI 653820,729533 FAM72B,FAM72A 195 FLFVDPELV 146850 PIK3R6 196 ILVDWLVQV 9133 CCNB2 SEQ ID No. Sequence GenelD(s) Official Gene Symbol(s) 197 VLLNEILEQV 64151 NCAPG 198 KIQEILTQV 10643 IGF2BP3 The present invention furthermore generally s to the peptides ing to the present invention for use in the treatment of proliferative diseases, such as, for example, bile duct cancer, brain cancer, breast , chronic lymphocytic leukemia, colon or rectum , esophageal cancer, gallbladder , liver cancer, melanoma, non-hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, small cell lung cancer, urinary bladder cancer, uterine cancer. ularly preferred are the peptides — alone or in combination - according to the present invention selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 188. More preferred are the es — alone or in combination - selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 100 (see Table 1), and their uses in the immunotherapy of AML, bile duct , brain cancer, breast cancer, chronic lymphocytic leukemia, colon or rectum cancer, esophageal cancer, gallbladder cancer, liver cancer, melanoma, non-hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, small cell lung , urinary bladder cancer, uterine cancer, and preferably AML.

As shown in the following Table 4, many of the peptides according to the present invention are also found on other tumor types and can, thus, also be used in the immunotherapy of other indications. Also referto Figure 1 and Example 1.

Table 4: Peptides ing to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases. The table shows for selected peptides on which onal tumor types they were found and either over- presented on more than 5% of the measured tumor s, or presented on more than 5% of the measured tumor samples with a ratio of geometric means tumor vs _ 23 _ normal tissues being larger than 3. Over-presentation is d as higher presentation on the tumor sample as compared to the normal sample with highest presentation.

Normal tissues against which over-presentation was tested were: adipose tissue, adrenal gland, artery, blood cells, bone marrow, brain, central nerve, colon, duodenum, esophagus, eye, gallbladder, heart, , liver, lung, lymph node, pancreas, parathyroid gland, peripheral nerve, peritoneum, pituitary gland, pleura, rectum, salivary gland, skeletal muscle, skin, small intestine, , stomach, thyroid gland, trachea, ureter, urinary bladder, vein.

SEQ ID No. Sequence Other relevant organs / diseases Melanoma, Esophageal Cancer, Urinary 1 LLDSAVYYL bladder , Uterine Cancer, adder Cancer, Bile Duct Cancer 6 GLAEVLLAA NHL, Uterine Cancer 7 LLWGDIMEL NHL 8 LLWPGAALLV NHL 9 SLLAYLEQA BRCA VlLDPVHSV SCLC, NHL, BRCA, Uterine Cancer 13 ALVPGVTQV CLL, NHL, Melanoma, Uterine Cancer 14 ALWWGTITL CLL, NHL NHL, Urinary bladder cancer, Gallbladder FIDEEVEDMYL Cancer, Bile Duct Cancer NHL, Gallbladder Cancer, Bile Duct GLDAFLLEL Cancer 23 GLLQPPVRIV HCC 24 GLVELLNRV NHL 27 KANPALYVL CLL 28 TPL Melanoma 29 RLGPSVVGL ma 31 PMV Urinary bladder cancer 32 SLSDRSWHL Brain Cancer 33 TIMNQEKLAKL CRC, NHL, Uterine Cancer 34 TLSPWSFLI NSCLC, e Cancer VLFEHAVGYAL CRC, CLL, NHL, BRCA, Melanoma, Urinary bladder cancer 36 VLGPSPSSV BRCA, ma 37 VVAPAPVVEAV PC 43 AMLDFVSSL NHL 45 FLFTDVLLM NHL 49 llDDNHAlV Melanoma _ 24 _ SEQ ID No. Sequence Other relevant organs / diseases 52 IVDPVDSTL Melanoma CLL, Gallbladder Cancer, Bile Duct 56 SAL Cancer 75 VLAPLGFTL BRCA 78 CHL Melanoma 79 YLMDPDTFTF SCLC 80 YLTEALQSI CLL, NHL Brain Cancer, HCC, PC, NHL, BRCA, Melanoma, Eso ha eal Cancer, Urinary 82 LLPDNFIAA bladder cancer, pUtegrline , Gallbladder Cancer, Bile Duct Cancer 83 GLGAGVAEAV CLL HCC, NHL, BRCA, Urinary bladder 84 GLLGSVLTI cancer, Uterine Cancer 86 HLLGDPMANV SCLC, 0C 90 LLLDTVTSI Brain Cancer, BRCA 93 SLINDVLAE CLL 94 SLLEFAQYL NHL Melanoma, Eso ha eal Cancer, 103 ALGDFGIRL Gallbladder Canpcerngile Duct Cancer 104 ALIPETTTL NHL, ma 105 GVFALVTAV NHL, BRCA, Melanoma, Uterine Cancer 106 ALLEELERSTL NHL 107 ALLGMLPLL CLL, NHL 108 ELEMNSDLKAQL Uterine Cancer 112 ILRDALNQA RCC, CLL, NHL, Melanoma 113 NLQSEVEGV PC, CLL 114 RLAQEAAQV RCC, OC, Uterine Cancer 115 SLPDLTTPL CLL, NHL, BRCA, Melanoma RCC, Melanoma, Gallbladder Cancer, 116 TILEILPEL Bile Duct Cancer 117 TILPTILFL CLL 118 TLLTVLTQA CLL, NHL 119 TLTDELAAL adder Cancer, Bile Duct Cancer 120 VIQDLWSV Melanoma, Uterine Cancer 121 VLQAGQYGV CLL, NHL, Uterine Cancer 122 VLL CLL, NHL, Uterine Cancer 123 PTL Gallbladder Cancer, Bile Duct Cancer HCC, CLL, NHL, BRCA, Melanoma, 124 GLPELVIQL Urinary bladder , Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 125 GLFGYLVFL SCLC, PC _ 25 _ SEQ ID No. Sequence Other relevant organs / diseases CLL, NHL, Eso ha eal Cancer, 0C, 127 KHSALPQL Gallbladder Caficeg Bile Duct Cancer 128 NLSTKTEAV NHL, OC, Uterine Cancer 129 RMAVLNEQV SCLC, NHL 132 SLYPVLNFL Urinary bladder cancer, Uterine Cancer 133 LFL NHL 135 GIIDRIFQA CRC, HCC, BRCA 136 GLSSIETLL BRCA 137 ILAPLAWDL CLL, NHL, e Cancer NSCLC, HCC, CLL, NHL, Melanoma, 138 ILSDNLRQV Esophageal Cancer, Urinary bladder cancer 140 TLL CLL, Melanoma PC, CLL, Urinary r cancer, Uterine 141 GLLPPLRIPELL Cancer, Gallbladder Cancer, Bile Duct Cancer CRC, PC, CLL, NHL, BRCA, Eso ha eal 142 g GLSDGYGFTT Cancer, OC, Urinary bladder canger 143 YLLPHILW RCC, Melanoma 144 GLFMGLVLV NHL, BRCA, Melanoma SCLC, CLL, NHL, Melanoma, Uterine 148 FLYFEDHGL Cancer 149 VLV RCC, Brain , NHL, Melanoma 150 NEI CLL, NHL, Uterine Cancer 151 GLLPFPEVTL CLL, NHL 152 GLSNHIAAL PC, NHL SCLC, RCC, CRC, HCC, CLL, NHL, BRCA, Melanoma, Esophageal Cancer, 154 llADNllFL OC, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer RCC, CLL, NHL, Melanoma, Uterine 155 lLDLlQVFV Cancer NSCLC, CRC, NHL, BRCA, Melanoma, 157 ILTETQQGL Urinary r cancer 160 RLVPFLVFV CLL BRCA, Esophageal Cancer, Gallbladder 161 SLIGIAIAL Cancer, Bile Duct Cancer 162 SLLDFLTFA NHL 163 SLMIDLIEV Melanoma, 0C 166 VLFPLNLQL CLL 167 VLLDVALGL RCC, NHL, BRCA, Melanoma 168 VLLFETALL CLL, BRCA, Urinary bladder cancer _ 26 _ SEQ ID No. Sequence Other relevant organs / diseases 169 VLQDPIWLL CLL, NHL RCC, CLL, NHL, Melanoma, Uterine 170 IVTEVAVGV Cancer 172 KLLWGDIMEL Gallbladder Cancer, Bile Duct Cancer Brain Cancer, PrC, NHL, BRCA, 173 KMQETLVGL Melanoma, Esophageal Cancer, Gallbladder , Bile Duct Cancer 174 QYV NHL 183 SVIGVSPAV Brain Cancer, NHL, BRCA, ma 184 RMTDQEAIQDL NHL, ma SCLC, NHL, Gallbladder Cancer, Bile 185 RLIPIIVLL Duct Cancer 186 llLDEAHNV CLL, NHL, Melanoma, e Cancer SCLC, RCC, Brain Cancer, CLL, NHL, 187 MLPPPPLTA BRCA, ma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer SCLC, RCC, CRC, HCC, CLL, NHL, 188 RLLDFPTLL BRCA, OC NSCLC= non-small cell lung cancer, SCLC= small cell lung cancer, RCC= kidney cancer, CRC= colon or rectum cancer, HCC= liver cancer, PC= pancreatic cancer, PrC= prostate cancer, BRCA= breast cancer, OC= ovarian cancer, CLL= chronic lymphocytic leukemia, NHL= non-hodgkin lymphoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one ofSEQ ID No. 37, 82, 113, 125, 141, 142, and 152 for the — in one preferred embodiment combined - treatment of pancreatic .

Thus, another aspect of the t invention s to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 32, 82, 90, 149, 173, 183, and 187 for the — in one preferred embodiment combined - treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide ing to the present invention according to any one of SEQ ID No. 1, 15, 20, 56, 82, 103, 116, 119, 123, 124, 127, 141, 154, 161, 172, 173, 185, and 187 for the — in one preferred embodiment combined - treatment of gallbladder and bile duct cancer.

Thus, r aspect of the present invention relates to the use of at least one peptide according to the t invention according to any one of SEQ ID No. 9, 10, 35, 36, 75, 82, 84, 90, 105, 115, 124, 135,136, 142, 144, 154, 157, 161, 167, 168,173, 183, 187, and 188 for the — in one preferred embodiment combined - treatment of breast .

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 13, 14, 27, 35, 56,80,83,93,107,112,113,115,117,118,121,122,124,127,137,138,140,141, 142, 148, 150, 151, 154, 155, 160, 166, 168, 169, 170, 186, 187, and 188 for the — in one preferred embodiment combined - ent of chronic lymphocytic leukemia.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present ion according to any one of SEQ ID No. 33, 35, 135, 142, 154, 157, and 188 for the — in one red embodiment combined - treatment of colorectal cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present ion according to any one of SEQ ID No. 1, 82, 103, 127, 138, 142, 154, 161, 173, and 187 for the — in one preferred embodiment combined - treatment of esophageal .

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 23, 82, 84, 124, 135, 138, 154, and 188 for the — in one preferred embodiment combined - treatment of liver cancer. _ 28 _ Thus, another aspect of the t invention relates to the use of at least one peptide ing to the present invention according to any one of SEQ ID No. 1, 13, 28, 29, 35, 36, 49, 52, 78, 82, 103, 104, 105, 112, 115, 116, 120, 124, 138, 140, 143, 144, 148, 149, 154, 155, 157, 163, 167, 170, 173, 183, 184, 186, and 187 for the — in one preferred embodiment combined - treatment of melanoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the t invention according to any one of SEQ ID No. 6, 7, 8, 10, 13, 14, 15, 20, 24, 33, 35,43, 45, 80, 82, 84, 94, 104, 105, 106, 107, 112, 115, 118, 121, 4,127,128,129,133,137,138,142,144,148,149,150,151,152,154,155, 157, 162, 167, 169, 170, 173, 174, 183, 184, 185, 186, 187, and 188 for the — in one red embodiment combined - treatment of non-hodgkin lymphoma.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 34, 138, and 157 for the — in one preferred embodiment combined - treatment of all cell lung cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide ing to the present invention according to any one of SEQ ID No. 86, 114, 127, 128, 142, 154, 163, and 188 for the — in one preferred embodiment combined - treatment of ovarian cancer.

Thus, another aspect of the present invention relates to the use of the peptide according to the present invention according to SEQ ID No. 173 for the treatment of prostate cancer.

Thus, another aspect of the present invention s to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 112, 114, 116, 143, 149, 154, 155, 167, 170, 187, and 188 for the — in one preferred embodiment combined - treatment of kidney cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 10, 79, 86, 125, 129, 148, 154, 185, 187, and 188 for the — in one preferred embodiment ed - treatment of small cell lung cancer.

Thus, r aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 1, 15, 31, 35, 82, 84, 124, 132, 138, 141, 142, 154, 157, 168, and 187 for the — in one preferred embodiment combined - ent of urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of at least one peptide according to the present invention according to any one of SEQ ID No. 1, 6, 10, 13, 33, 34,82,84,105,108,114,120,121,122,124,128,132,137,141,148,150,154,155, 170, 186, and 187 for the — in one red embodiment combined - treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of the peptides according to the present invention for the - preferably combined - treatment of a proliferative disease selected from the group of AML, bile duct cancer, brain cancer, breast cancer, chronic cytic ia, colon or rectum cancer, esophageal cancer, gallbladder cancer, liver cancer, melanoma, non-hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate , kidney cancer, small cell lung cancer, urinary bladder cancer, uterine .

The present invention furthermore s to peptides according to the present invention that have the ability to bind to a molecule of the human major ompatibility complex (MHC) class-l or - in an elongated form, such as a length-variant - MHC class -II.

WO 74645 _ 30 _ The present invention further relates to the peptides ing to the present invention wherein said peptides (each) consist or consist essentially of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 188.

The present invention further relates to the peptides according to the present invention, wherein said peptide is modified and/or includes non-peptide bonds.

The present ion further relates to the peptides according to the present invention, wherein said peptide is part of a fusion protein, in particular fused to the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii), or fused to (or into the sequence of) an antibody, such as, for example, an antibody that is ic for dendritic cells.

The present invention further relates to a nucleic acid, encoding the peptides according to the present invention. The present invention further relates to the nucleic acid according to the present invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present ion further relates to an expression vector capable of expressing and/or expressing a nucleic acid according to the present invention.

The present invention further s to a peptide according to the present invention, a nucleic acid according to the t invention or an expression vector according to the present invention for use in the treatment of diseases and in medicine, in particular in the ent of cancer.

The present invention r s to antibodies that are ic against the peptides according to the present invention or complexes of said peptides according to the t invention with MHC, and methods of making these.

WO 74645 _ 31 _ The present invention further relates to T-cell receptors (TCRs), in particular soluble TCR ) and cloned TCRs engineered into gous or allogeneic T cells, and methods of making these, as well as NK cells or other cells bearing said TCR or cross- reacting with said TCRs.

The dies and TCRs are additional embodiments of the immunotherapeutic use of the peptides according to the invention at hand.

The present invention further relates to a host cell comprising a nucleic acid according to the present invention or an expression vector as bed before. The present invention further relates to the host cell according to the present ion that is an antigen presenting cell, and preferably is a dendritic cell.

The present ion further relates to a method for producing a peptide according to the present invention, said method comprising culturing the host cell according to the present invention, and isolating the peptide from said host cell or its culture medium.

The t invention further relates to said method according to the present invention, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable n-presenting cell or artificial antigen-presenting cell by ting a sufficient amount of the antigen with an antigen-presenting cell.

The present invention further s to the method according to the present invention, wherein the antigen-presenting cell comprises an expression vector capable of expressing or expressing said peptide containing SEQ ID No. 1 to SEQ ID No.: 188, preferably containing SEQ ID No. 1 to SEQ ID No. 100, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced by the method according to the present invention, wherein said T cell selectively recognizes a cell which expresses a polypeptide sing an amino acid sequence according to the t invention.

The present invention further relates to a method of killing target cells in a patient which target cells aberrantly express a polypeptide comprising any amino acid sequence according to the present invention, the method comprising administering to the patient an effective number ofT cells as produced according to the present invention.

The t invention further relates to the use of any peptide as described, the nucleic acid according to the present invention, the expression vector according to the present invention, the cell according to the t invention, the activated T cyte, the T cell or or the antibody or other peptide- and/or peptide-MHC-binding molecules according to the present invention as a medicament or in the cture of a ment. ably, said medicament is active against cancer.

Preferably, said medicament is a cellular therapy, a vaccine or a protein based on a soluble TCR or dy.

The present invention further relates to a use according to the present invention, wherein said cancer cells are AML, bile duct cancer, brain cancer, breast cancer, chronic lymphocytic ia, colon or rectum cancer, geal cancer, gallbladder cancer, liver cancer, melanoma, non-hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, small cell lung cancer, urinary bladder cancer, uterine cancer, and preferably AML cells.

The present invention further relates to biomarkers based on the peptides according to the present invention, herein called "targets" that can be used in the diagnosis of , preferably AML. The marker can be over-presentation of the peptide(s) themselves, or over-expression of the corresponding gene(s). The markers may also be used to predict the probability of success of a treatment, preferably an therapy, and most preferred an immunotherapy targeting the same target that is identified by the _ 33 _ biomarker. For example, an antibody or e TCR can be used to stain sections of the tumor to detect the presence of a peptide of interest in complex with MHC.

Optionally the antibody s a further effector function such as an immune stimulating domain or toxin.

The present invention also relates to the use of these novel targets in the context of cancer treatment.

DETAILED DESCRIPTION OF THE INVENTION Stimulation of an immune response is dependent upon the presence of antigens ized as foreign by the host immune system. The discovery of the existence of tumor associated antigens has raised the possibility of using a host's immune system to intervene in tumor . Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. The isolation of T-cells from tumor-infiltrating cell populations or from eral blood suggests that such cells play an ant role in natural immune defense against . CD8—positive T-cells in particular, which recognize class I molecules of the major histocompatibility complex (MHC)—bearing peptides of usually 8 to 10 amino acid residues d from proteins or defect ribosomal products (DRIPS) located in the cytosol, play an ant role in this response. The MHC-molecules of the human are also designated as human leukocyte- antigens (HLA).

As used herein and except as noted otherwise all terms are defined as given below. _ 34 _ The term "T-cell response" means the specific proliferation and activation of effector functions d by a peptide in vitro or in vivo. For MHC class I restricted cytotoxic T cells, effector functions may be lysis of peptide-pulsed, peptide-precursor pulsed or naturally peptide-presenting target cells, secretion of cytokines, preferably Interferongamma , TNF-alpha, or lL-2 induced by peptide, secretion of effector molecules, ably granzymes or perforins induced by peptide, or degranulation.

The term "peptide" is used herein to designate a series of amino acid es, connected one to the other typically by e bonds between the amino and carbonyl groups of the adjacent amino acids. The peptides are ably 9 amino acids in length, but can be as short as 8 amino acids in length, and as long as 10, 11, 12, 13 or 14 or longer, and in case of MHC class II peptides (elongated variants of the peptides of the invention) they can be as long as 15, 16, 17, 18, 19 or 20 or more amino acids in length.

Furthermore, the term "peptide" shall include salts of a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. Preferably, the salts are pharmaceutical acceptable salts of the peptides, such as, for example, the chloride or acetate uoroacetate) salts. It has to be noted that the salts of the peptides according to the present invention differ substantially from the peptides in their state(s) in vivo, as the peptides are not salts in vivo.

The term "peptide" shall also include "oligopeptide". The term "oligopeptide" is used herein to designate a series of amino acid es, connected one to the other typically by e bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the oligopeptide is not critical to the invention, as long as the correct epitope or epitopes are maintained therein. The oligopeptides are lly less than about 30 amino acid residues in length, and r than about 15 amino acids in length. _ 35 _ The term "polypeptide" designates a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the polypeptide is not critical to the invention as long as the correct epitopes are maintained. In contrast to the terms peptide or oligopeptide, the term polypeptide is meant to refer to molecules containing more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such a molecule is "immunogenic" (and thus is an "immunogen" within the present invention), if it is capable of inducing an immune response. In the case of the present invention, genicity is more specifically defined as the ability to induce a T-cell response. Thus, an ogen" would be a molecule that is capable of inducing an immune response, and in the case of the present invention, a molecule capable of inducing a T-cell response.

In another aspect, the immunogen can be the peptide, the complex of the peptide with MHC, oligopeptide, and/or protein that is used to raise specific dies or TCRs against it.

A class I T cell "epitope" requires a short peptide that is bound to a class I MHC receptor, forming a ternary complex (MHC class I alpha chain, betamicroglobulin, and peptide) that can be recognized by a T cell bearing a matching T-cell receptor binding to the MHC/peptide complex with appropriate ty. Peptides binding to MHC class I molecules are typically 8-14 amino acids in length, and most typically 9 amino acids in length.

In humans there are three ent genetic loci that encode MHC class I les (the MHC-molecules of the human are also designated human leukocyte antigens : HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*O7 are examples of different MHC class I alleles that can be sed from these loci.

Table 5: Expression ncies F of HLA-A*02 and HLA-A*24 and the most frequent HLA-DR pes. Frequencies are deduced from haplotype frequencies Gf within the American population adapted from Mori et al. (Mori et al., 1997) employing the Hardy- Weinberg formula F = 1 — (1-Gf)2. Combinations of A*02 or A*24 with certain HLA-DR alleles might be enriched or less frequent than expected from their single frequencies due to linkage disequilibrium. For details refer to Chanock et al. ck et al., 2004).

Allele Population ated phenotype from allele frequency A*02 ian (North America) 49.1% A*02 n American (North America) 34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American) 48.3% DR1 Caucasian (North America) 19.4% DR2 Caucasian (North America) 28.2% DR3 Caucasian (North a) 20.6% DR4 Caucasian (North America) 30.7% DR5 Caucasian (North America) 23.3% DR6 Caucasian (North America) 26.7% DR7 Caucasian (North America) 24.8% DR8 Caucasian (North America) 5.7% DR9 Caucasian (North America) 2.1% DR1 African (North) American 13.20% DR2 African (North) American 29.80% DR3 African (North) American 24.80% DR4 African (North) American 11.10% DR5 African (North) American 31.10% DR6 African (North) American 33.70% DR7 African (North) American 19.20% DR8 African (North) American 12.10% DR9 n (North) American 5.80% DR1 Asian (North) an 6.80% DR2 Asian (North) American 33.80% DR3 Asian (North) an 9.20% DR4 Asian (North) American 28.60% DR5 Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7 Asian (North) an 13.40% DR8 Asian ) American 12.70% DR9 Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2 Latin (North) American 21.20% _ 37 _ Allele Population Calculated phenotype from allele frequency DR3 Latin (North) American 15.20% DR4 Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6 Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8 Latin (North) American 18.60% DR9 Latin (North) American 2.10% A*24 Philippines 65% A*24 Russia Nenets 61% A*24:02 Japan 59% A*24 Malaysia 58% A*24:02 Philippines 54% A*24 India 47% A*24 South Korea 40% A*24 Sri Lanka 37% A*24 China 32% A*24:02 India 29% A*24 Australia West 22% A*24 USA 22% A*24 Russia Samara 20% A*24 South America 20% A*24 Europe 18% The peptides of the invention, preferably when included into a vaccine of the invention as bed herein bind to A*02. A vaccine may also include pan-binding MHC class II peptides. Therefore, the e of the invention can be used to treat cancer in patients that are A*02 positive, whereas no selection for MHC class II allotypes is necessary due to the nding nature of these es. |f A*02 peptides of the invention are combined with peptides binding to another allele, for example A*24, a higher percentage of any patient population can be treated ed with addressing either MHC class I allele alone. While in most populations less than 50% of patients could be addressed by either allele alone, a vaccine sing HLA-A*24 and HLA-A*02 epitopes can treat at least 60% of patients in any relevant tion. Specifically, the ing percentages of patients will be positive for _ 38 _ at least one of these alleles in various regions: USA 61%, Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculated from lelefrequencies.net).

In a red embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, eptide, or ptide may be naturally occurring or they may be synthetically constructed. Generally, DNA segments encoding the peptides, polypeptides, and proteins of this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene that is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.

As used herein the term "a nucleotide coding for (or encoding) a peptide" refers to a nucleotide sequence coding for the peptide including artificial (man-made) start and stop codons compatible for the biological system the sequence is to be expressed by, for example, a dendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes both single stranded and double stranded nucleic acid. Thus, for example for DNA, the specific sequence, unless the context indicates otherwise, refers to the single strand DNA of such ce, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.

The term "coding region" refers to that n of a gene which either naturally or normally codes for the expression product of that gene in its l genomic nment, i.e., the region coding in vivo for the native expression product of the gene. _ 3g _ The coding region can be derived from a non-mutated ("normal"), mutated or d gene, or can even be derived from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.

The term "expression product" means the polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding lents resulting from genetic code degeneracy and thus coding for the same amino acid(s).

The term ent", when referring to a coding sequence, means a portion of DNA comprising less than the complete coding region, whose expression product s ially the same biological function or activity as the sion product of the complete coding region.

The term "DNA segment" refers to a DNA r, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide ces by standard biochemical methods, for example, by using a cloning vector. Such segments are provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are lly present in eukaryotic genes. Sequences of non-translated DNA may be t downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

The term "primer" means a short nucleic acid sequence that can be paired with one strand of DNA and provides a free 3'-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.

The term "promoter" means a region of DNA involved in binding of RNA polymerase to initiate transcription.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment, if it is naturally occurring). For example, a lly- occurring cleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The cleotides, and recombinant or immunogenic ptides, disclosed in accordance with the present invention may also be in "purified" form. The term "purified" does not require absolute purity; rather, it is intended as a relative definition, and can e preparations that are highly purified or ations that are only partially purified, as those terms are understood by those of skill in the relevant art. For example, dual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. Purification of starting material or natural material to at least one order of magnitude, ably two or three , and more preferably four or five orders of magnitude is expressly contemplated. Furthermore, a claimed polypeptide which has a purity of preferably 99.999%, or at least 99.99% or 99.9%; and even bly 99% by weight or greater is expressly encompassed.

The nucleic acids and polypeptide expression products sed according to the present invention, as well as expression vectors containing such c acids and/or such polypeptides, may be in "enriched form". As used herein, the term "enriched" means that the concentration of the al is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 5%, %, and 20% by weight are also contemplated. The sequences, constructs, vectors, clones, and other materials comprising the present invention can advantageously be in enriched or isolated form. The term "active fragment" means a fragment, usually of a peptide, polypeptide or nucleic acid sequence, that generates an immune response (i.e., has immunogenic activity) when administered, alone or optionally with a suitable WO 74645 adjuvant or in a vector, to an animal, such as a mammal, for example, a rabbit or a mouse, and also including a human, such immune response taking the form of stimulating a T-cell response within the recipient animal, such as a human. Alternatively, the "active fragment" may also be used to induce a T-cell response in vitro.

As used herein, the terms "portion , segment" and "fragment", when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were ted to ent with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent ns, segments or fragments of the starting polypeptide. When used in relation to polynucleotides, these terms refer to the products produced by treatment of said polynucleotides with any of the endonucleases.

In ance with the present invention, the term "percent identity" or "percent identical", when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the "Reference Sequence"). The percent identity is then determined according to the following formula: percent identity = 100 [1 -(C/R)] wherein C is the number of differences between the Reference Sequence and the ed Sequence over the length of alignment between the Reference Sequence and the ed ce, wherein (i) each base or amino acid in the nce Sequence that does not have a ponding d base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the nce Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference and (iiii) the alignment has to start at position 1 of the aligned sequences; and R is the number of bases or amino acids in the Reference ce over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent ty to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified t identity.

As mentioned above, the present invention thus provides a e comprising a ce that is selected from the group of consisting of SEQ ID NO: 1 to SEQ ID NO: 188 or a variant thereof which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 188, or a variant thereof that will induce T cells cross-reacting with said peptide. The peptides of the invention have the ability to bind to a molecule of the human major ompatibility complex (MHC) class-I or elongated versions of said peptides to class In the present invention, the term "homologous" refers to the degree of identity (see percent identity above) between sequences of two amino acid sequences, i.e. peptide or polypeptide sequences. The aforementioned "homology" is ined by comparing two sequences aligned under l conditions over the ces to be compared.

Such a sequence homology can be calculated by creating an alignment using, for example, the ClustalW algorithm. Commonly available sequence analysis software, more specifically, Vector NTI, GENETYX or other tools are provided by public databases.

A person skilled in the art will be able to assess, whether T cells induced by a t of a specific peptide will be able to cross-react with the peptide itself (Appay et al., 2006; etti et al., 2006; Fong et al., 2001; Zaremba et al., 1997).

By a nt" of the given amino acid sequence the inventors mean that the side chains of, for example, one or two of the amino acid residues are altered (for example by replacing them with the side chain of another naturally occurring amino acid residue or some other side chain) such that the e is still able to bind to an HLA molecule in substantially the same way as a peptide consisting of the given amino acid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 188. For example, a peptide may be modified so that it at least maintains, if not improves, the ability to interact with and bind to the binding groove of a suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it at least maintains, if not improves, the y to bind to the TCR of activated T cells.

These T cells can subsequently cross-react with cells and kill cells that express a polypeptide that contains the natural amino acid sequence of the cognate peptide as defined in the s of the invention. As can be derived from the ific literature and databases (Rammensee et al., 1999; Godkin et al., 1997), certain positions of HLA binding peptides are typically anchor residues forming a core ce fitting to the binding motif of the HLA receptor, which is defined by polar, electrophysical, hydrophobic and spatial properties of the polypeptide chains constituting the binding groove. Thus, one skilled in the art would be able to modify the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO 188, by maintaining the known anchor residues, and would be able to determine r such variants maintain the ability to bind MHC class I or II molecules. The variants of the present invention retain the ability to bind to the TCR of activated T cells, which can subsequently cross-react with and kill cells that express a polypeptide containing the l amino acid sequence of the cognate peptide as defined in the aspects of the invention.

The al ified) peptides as disclosed herein can be modified by the substitution of one or more residues at different, possibly selective, sites within the peptide chain, if not otherwise stated. Preferably those substitutions are located at the end of the amino acid chain. Such substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical , such as where leucine is replaced by cine. In studies of ce ions in families of naturally occurring homologous proteins, n amino acid substitutions are more often tolerated than others, and these are often show correlation with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, and such is the basis for defining "conservative substitutions." Conservative substitutions are herein defined as exchanges within one of the following five groups: Group 1-small aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar, positively charged residues (His, Arg, Lys); Group 4-large, aliphatic, nonpolar residues (Met, Leu, lle, Val, Cys); and Group 5-large, aromatic residues (Phe, Tyr, Trp).

Less vative substitutions might involve the replacement of one amino acid by another that has r characteristics but is somewhat different in size, such as replacement of an alanine by an isoleucine residue. Highly non-conservative replacements might involve tuting an acidic amino acid for one that is polar, or even for one that is basic in character. Such "radical" substitutions cannot, however, be dismissed as potentially ctive since chemical effects are not totally table and radical tutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles.

Of course, such substitutions may e structures other than the common L-amino acids. Thus, D-amino acids might be substituted for the L-amino acids commonly found in the antigenic peptides of the invention and yet still be encompassed by the disclosure herein. In addition, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids) may also be used for substitution purposes to produce immunogens and genic polypeptides according to the t invenfion. lf substitutions at more than one position are found to result in a peptide with substantially equivalent or greater antigenic activity as defined below, then combinations of those substitutions will be tested to determine if the combined substitutions result in additive or synergistic effects on the antigenicity of the peptide. At most, no more than 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicated herein can have one or two non-anchor amino acids (see below regarding the anchor motif) exchanged without that the ability to bind to a molecule of the human major histocompatibility x (MHC) class-l or —II is substantially changed or is negatively affected, when ed to the non-modified peptide. In another embodiment, in a peptide consisting essentially of the amino acid sequence as indicated herein, one or two amino acids can be exchanged with their conservative exchange partners (see herein below) without that the ability to bind to a molecule of the human major histocompatibility x (MHC) class-l or —II is substantially changed, or is negatively ed, when compared to the non-modified peptide.

The amino acid residues that do not substantially contribute to interactions with the T- cell receptor can be ed by replacement with other amino acid whose oration does not substantially affect T-cell reactivity and does not eliminate binding to the relevant MHC. Thus, apart from the proviso given, the peptide of the invention may be any e (by which term the ors include oligopeptide or polypeptide), which includes the amino acid sequences or a portion or variant thereof as given.

Table 6: Variants and motif of the peptides according to SEQ ID NO: 1, 9, and 82 Position 1 2 3 4 5 6 7 8 g SEQID NO.1 L L D S A V Y Y L Variants A on 1 N 00 4; 01 CD \I CD <0 Position 1 co 4> 01 c» \I oo SEQ ID NO. 9 S I—NQQQQ—i—l—l—l<<<<>>>>§§§§ Variants |——<>©<—> <<<<>>>>§§§§ < Position 1 Position 1 SEQ ID NO. 82 L .0 D Z ‘n > Variants l——©|——< QQQQ—I—I—I—I<<<<>>>>§§§§ < Longer ated) peptides may also be suitable. It is possible that MHC class I epitopes, although usually between 8 and 11 amino acids long, are generated by peptide processing from longer es or proteins that include the actual epitope. It is preferred that the residues that flank the actual epitope are residues that do not substantially affect proteolytic ge necessary to expose the actual epitope during processing.

The peptides of the invention can be elongated by up to four amino acids, that is 1, 2, 3 or 4 amino acids can be added to either end in any ation n 4:0 and 0:4.

Combinations of the elongations according to the invention can be found in Table 7.

Table 7: Combinations of the elongations of peptides of the invention C-terminus N-terminus o—xmoohzo—xmooh O Oorior2 Oor10r20r3 Oor1 or20r3or4 nus C-terminus Oorior2 Oor10r20r3 Oor1 or20r3or4 The amino acids for the elongation/extension can be the peptides of the al sequence of the protein or any other amino acid(s). The elongation can be used to enhance the stability or solubility of the es.

Thus, the epitopes of the present invention may be identical to naturally occurring tumor-associated or tumor-specific epitopes or may include epitopes that differ by no more than four residues from the reference peptide, as long as they have substantially identical antigenic activity.

In an ative embodiment, the peptide is elongated on either or both sides by more than 4 amino acids, preferably to a total length of up to 30 amino acids. This may lead to MHC class II binding peptides. Binding to MHC class II can be tested by methods known in the art.

Accordingly, the present ion provides peptides and variants of MHC class I es, wherein the peptide or variant has an overall length of between 8 and 100, preferably between 8 and 30, and most preferred between 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in case of the elongated class II binding peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

Of , the e or variant according to the present ion will have the ability to bind to a molecule of the human major ompatibility complex (MHC) class I or II.

Binding of a peptide or a variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to the present invention are tested against the substituted peptides, the peptide concentration at which the substituted peptides achieve half the maximal increase in lysis relative to background is no more than about 1 mM, preferably no more than about 1 pM, more preferably no more than about 1 nM, and still more preferably no more than about 100 pM, and most preferably no more than about 10 pM. It is also red that the substituted peptide be recognized by T cells from more than one individual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptide consists or consists essentially of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 188.

"Consisting essentially of" shall mean that a peptide according to the present invention, in addition to the sequence according to any of SEQ ID NO: 1 to SEQ ID NO 188 or a variant thereof ns additional N- and/or C-terminally d stretches of amino acids that are not necessarily forming part of the peptide that functions as an epitope for MHC molecules epitope.

Nevertheless, these stretches can be important to e an efficient introduction of the e according to the present invention into the cells. In one embodiment of the present invention, the peptide is part of a fusion protein which comprises, for example, the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33, in the following "li") as derived from the NCBI, GenBank Accession number X00497. In other fusions, the peptides of the present invention can be fused to an antibody as described herein, or a functional part thereof, in particular into a sequence of an antibody, so as to be specifically targeted by said antibody, or, for example, to or into an antibody that is specific for dendritic cells as described herein.

In addition, the peptide or t may be modified further to e stability and/or binding to MHC molecules in order to elicit a stronger immune response. s for such an zation of a peptide sequence are well known in the art and include, for e, the introduction of reverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide H-) linkages but the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using s known in the art, for example such as those bed in Meziere et al (1997) (Meziere et al., 1997), incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al. (Meziere et al., 1997) show that for MHC binding and T helper cell responses, these pseudopeptides are useful. Retro-inverse peptides, which contain NH-CO bonds d of CO-NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, -CH2-NH, -CH28-, -CH20H2-, -CH=CH-, -COCH2-, - CH(OH)CH2-, and -CH280-. US 4,897,445 provides a method for the solid phase synthesis of ptide bonds (-CH2-NH) in polypeptide chains which involves polypeptides synthesized by standard procedures and the non-peptide bond synthesized by reacting an amino aldehyde and an amino acid in the presence of NaCNBH3.

Peptides comprising the sequences described above may be synthesized with additional al groups present at their amino and/or carboxy termini, to enhance the stability, bioavailability, and/or affinity of the es. For example, hydrophobic groups such as enzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides' amino termini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter their steric configuration. For example, the D-isomer of one or more of the amino acid residues of the peptide may be used, rather than the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the invention may be tuted by one of the well-known non-naturally occurring amino acid residues. Alterations such as these may sen/e to increase the stability, bioavailability and/or binding action of the peptides of the invenfion.

Similarly, a peptide or variant of the ion may be modified chemically by reacting specific amino acids either before or after synthesis of the peptide. Examples for such modifications are well known in the art and are summarized e.g. in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which is incorporated herein by reference. Chemical modification of amino acids es but is not limited to, cation by ion, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups and sulphydryl modification by mic acid oxidation of cysteine to cysteic acid, formation of mercurial tives, formation of mixed disulphides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide and carbamoylation with cyanate at alkaline pH, although without limitation thereto. In this regard, the d person is referred to Chapter 15 of Current Protocols ln Protein _ 52 _ e, Eds. Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995) for more extensive methodology relating to chemical modification of proteins.

Briefly, modification of e.g. arginyl residues in proteins is often based on the on of vicinal dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2- cyclohexanedione to form an adduct. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without itant modification of other nucleophilic sites such as lysine and histidine. As a result, a large number of reagents are available for the modification of cysteine. The websites of companies such as Sigma-Aldrich (http://www.sigma-aldrich.com) provide information on specific reagents.

Selective ion of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during the heat treatment of rmaceuticals. Woodward’s Reagent K may be used to modify specific glutamic acid residues. N-(3- (dimethylamino)propyl)-N’-ethylcarbodiimide can be used to form molecular crosslinks between a lysine residue and a glutamic acid residue. For e, diethylpyrocarbonate is a reagent for the modification of histidyl residues in proteins.

Histidine can also be modified using oxynonenal. The reaction of lysine residues and other d-amino groups is, for example, useful in binding of peptides to surfaces or the cross-linking of ns/peptides. Lysine is the site of attachment of poly(ethylene)glycol and the major site of modification in the glycosylation of proteins.

Methionine residues in proteins can be modified with e.g. iodoacetamide, bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosyl residues. Cross-linking via the formation of dityrosine can be lished with en peroxide/copper ions. _ 53 _ Recent s on the modification of phan have used N-bromosuccinimide, 2- hydroxynitrobenzyl bromide or 3-bromomethyl(2—nitrophenylmercapto)-3H- indole (BPNS-skatole).

Successful modification of eutic proteins and peptides with PEG is often associated with an extension of circulatory half-life while cross-linking of proteins with glutaraldehyde, polyethylene glycol diacrylate and formaldehyde is used for the preparation of hydrogels. Chemical modification of allergens for immunotherapy is often achieved by carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includes non-peptide bonds is a preferred embodiment of the invention. Generally, peptides and variants (at least those containing peptide linkages between amino acid residues) may be synthesized by the Fmoc-polyamide mode of phase peptide synthesis as disclosed by Lukas et al.

(Lukas et al., 1981) and by references as cited therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is done using 20% dine in N, N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where ine or asparagine are C-terminal es, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl- acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalizing agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid tive. All amino acid derivatives are added as their preformed rical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N, N- ohexyl-carbodiimide/1hydroxybenzotriazole ed coupling procedure. All coupling and deprotection ons are monitored using ninhydrin, trinitrobenzene nic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by ent with 95% trifluoroacetic acid containing a 50 % scavenger mix.

Scavengers commonly used include ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesized. Also a combination of solid phase and solution phase methodologies for the synthesis of peptides is possible (see, for example, (Bruckdorfer et al., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent ation with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilization of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide sis are generally available from e.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of, techniques such as re- crystallization, size exclusion chromatography, ion-exchange tography, hydrophobic interaction chromatography and (usually) reverse-phase high performance liquid chromatography using e.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer tography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid tography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESl-Q-TOF mass spectrometric analysis.

In order to select over-presented peptides, a tation e is calculated showing the median sample presentation as well as replicate variation. The e juxtaposes samples of the tumor entity of interest to a baseline of normal tissue samples. Each of these profiles can then be consolidated into an over-presentation score by calculating _ 55 _ the p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015) adjusting for multiple testing by False Discovery Rate (Benjamini and Hochberg, 1995) (cf. Example 1, s 1).

For the identification and relative quantitation of HLA ligands by mass spectrometry, HLA molecules from shock-frozen tissue samples were purified and HLA-associated peptides were isolated. The ed peptides were separated and ces were identified by online nano-electrospray-ionization (nanoESl) liquid chromatography-mass spectrometry (LC-MS) experiments. The resulting peptide sequences were verified by comparison of the ntation pattern of natural tumor-associated peptides (TUMAPs) recorded from AML samples (N = 17 A*02—positive samples) with the fragmentation patterns of corresponding synthetic reference peptides of identical sequences. Since the peptides were directly identified as s of HLA les of primary tumors, these results provide direct evidence for the natural processing and presentation of the identified peptides on primary cancer tissue obtained from 16 AML patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for e, US 2013-0096016, which is hereby incorporated by reference in its entirety) allows the identification and selection of relevant over-presented peptide vaccine candidates based on direct relative quantitation of HLA-restricted peptide levels on cancer tissues in ison to several ent ncerous tissues and organs. This was achieved by the development of label-free differential tation using the acquired LC-MS data processed by a proprietary data analysis ne, combining algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, charge state deconvolution and normalization.

Presentation levels including error estimates for each peptide and sample were established. Peptides exclusively presented on tumor tissue and peptides over- ted in tumor versus non-cancerous tissues and organs have been identified.

WO 74645 _ 56 _ HLA-peptide complexes from AML tissue samples were purified and sociated peptides were isolated and analyzed by LC-MS (see examples). All TUMAPs contained in the present application were identified with this approach on primary AML samples confirming their presentation on primary AML.

TUMAPs identified on multiple AML and normal tissues were quantified using ion- counting of label-free LC-MS data. The method s that LC-MS signal areas of a peptide correlate with its abundance in the sample. All quantitative signals of a peptide in various LC-MS experiments were normalized based on central tendency, ed per sample and merged into a bar plot, called presentation profile. The presentation profile consolidates ent analysis methods like protein database search, al clustering, charge state deconvolution (decharging) and retention time alignment and ization.

Besides over-presentation of the peptide, mRNA expression of the underlying gene was tested. mRNA data were ed via RNASeq analyses of normal tissues and cancer tissues (cf. Example 2, Figure 2). An onal source of normal tissue data was a database of publicly available RNA expression data from around 3000 normal tissue s (Lonsdale, 2013). Peptides which are derived from proteins whose coding mRNA is highly expressed in cancer tissue, but very low or absent in vital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treating cancers/tumors, preferably AML that over- or exclusively present the peptides of the invention. These peptides were shown by mass spectrometry to be naturally presented by HLA molecules on primary human AML samples.

Many of the source gene/proteins (also designated "full-length proteins" or lying proteins") from which the peptides are derived were shown to be highly over-expressed in cancer compared with normal tissues — "normal s" in relation to this invention shall mean either healthy bone marrow and blood cells or other normal tissue cells, demonstrating a high degree of tumor association of the source genes (see e 2).

Moreover, the es themselves are strongly over-presented on tumor tissue — "tumor " in relation to this invention shall mean a sample from a t suffering from AML, but not on normal tissues (see Example 1).

HLA-bound es can be recognized by the immune system, specifically T lymphocytes. T cells can destroy the cells presenting the recognized HLA/peptide complex, e.g. AML cells presenting the derived peptides.

The peptides of the present invention have been shown to be e of ating T cell ses and/or are over-presented and thus can be used for the production of antibodies and/or TCRs, such as soluble TCRs, according to the present invention (see Example 3, e 4). Furthermore, the peptides when complexed with the respective MHC can be used for the production of antibodies and/or TCRs, in particular sTCRs, according to the present invention, as well. Respective methods are well known to the person of skill, and can be found in the respective literature as well. Thus, the peptides of the present invention are useful for generating an immune response in a patient by which tumor cells can be destroyed. An immune response in a patient can be induced by direct administration of the described es or suitable precursor substances (e.g. elongated peptides, proteins, or nucleic acids encoding these peptides) to the patient, ideally in combination with an agent enhancing the immunogenicity (i.e. an nt).

The immune response originating from such a therapeutic vaccination can be expected to be highly specific against tumor cells because the target peptides of the present invention are not presented on normal tissues in comparable copy numbers, preventing the risk of undesired autoimmune reactions against normal cells in the patient.

The present description r relates to T-cell receptors (TCRs) comprising an alpha chain and a beta chain ("alpha/beta TCRs"). Also provided are peptides according to the invention capable of binding to TCRs and antibodies when presented by an MHC molecule. The present description also relates to nucleic acids, vectors and host cells _ 58 _ for expressing TCRs and peptides of the present ption; and methods of using the same.

The term "T-cell receptor" (abbreviated TCR) refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta ptide chain (beta chain), wherein the heterodimeric receptor is capable of binding to a e antigen presented by an HLA molecule. The term also includes so-called delta TCRs.

In one embodiment the description es a method of producing a TCR as described herein, the method comprising culturing a host cell capable of expressing the TCR under conditions suitable to e expression of the TCR.

The description in another aspect relates to methods according to the description, wherein the antigen is loaded onto class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or artificial n-presenting cell by contacting a ient amount of the antigen with an antigen-presenting cell or the antigen is loaded onto class I or II MHC tetramers by tetramerizing the antigen/class | or II MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and delta chains of gamma/delta TCRs, are generally regarded as each having two "domains", namely variable and constant domains. The variable domain consists of a concatenation of variable region (V), and joining region (J). The variable domain may also include a leader region (L). Beta and delta chains may also include a diversity region (D). The alpha and beta constant domains may also include C-terminal transmembrane (TM) domains that anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term "TCR gamma variable domain" as used herein refers to the concatenation of the TCR gamma V (TRGV) region without leader region (L), and the TCR gamma J (TRGJ) region, and the term TCR gamma constant domain refers to the extracellular TRGC region, or to a inal truncated TRGC ce. Likewise the term "TCR delta variable domain" refers to the concatenation of the TCR delta V (TRDV) region without leader region (L) and the TCR delta D/J (TRDD/TRDJ) region, and the term "TCR delta constant domain" refers to the extracellular TRDC region, or to a C-terminal truncated TRDC sequence.

TCRs of the present description preferably bind to an peptide-HLA molecule complex with a binding affinity (KD) of about 100 uM or less, about 50 uM or less, about 25 uM or less, or about 10 uM or less. More red are high affinity TCRs having binding affinities of about 1 uM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less. Non-limiting examples of red binding affinity ranges for TCRs of the present invention e about 1 nM to about 10 nM; about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description, fic binding" and grammatical variants thereof are used to mean a TCR having a binding affinity (KD) for a peptide-HLA le complex of 100 uM or less.

Alpha/beta heterodimeric TCRs of the present description may have an introduced disulfide bond between their constant domains. Preferred TCRs of this type include those which have a TRAC constant domain sequence and a TRBC1 or TRBCZ constant domain sequence except that Thr 48 of TRAC and Ser 57 of TRBCl or TRBCZ are replaced by cysteine residues, the said cysteines forming a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBCZ nt domain sequence of the TCR.

With or without the introduced inter-chain bond ned above, alpha/beta hetero- dimeric TCRs of the present description may have a TRAC nt domain sequence and a TRBCl or TRBCZ constant domain sequence, and the TRAC constant domain sequence and the TR801 or TRBCZ constant domain sequence of the TCR may be _ 60 _ linked by the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBCi or TRBCZ.

TCRs of the present description may se a detectable label ed from the group consisting of a radionuclide, a fluorophore and biotin. TCRs of the present description may be conjugated to a therapeutically active agent, such as a radionuclide, a chemotherapeutic agent, or a toxin.

In an embodiment, a TCR of the present ption having at least one mutation in the alpha chain and/or having at least one mutation in the beta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCR alpha chain and/or TCR beta chain has a binding affinity for, and/or a g half-life for, a peptide- HLA molecule complex, which is at least double that of a TCR comprising the unmutated TCR alpha chain and/or ted TCR beta chain. Affinity-enhancement of tumor-specific TCRs, and its exploitation, relies on the existence of a window for optimal TCR affinities. The existence of such a window is based on observations that TCRs specific for -restricted pathogens have KD values that are generally about 10- fold lower when compared to TCRs specific for HLA-A2—restricted tumor-associated self-antigens. It is now known, although tumor antigens have the potential to be immunogenic, because tumors arise from the individual’s own cells only mutated proteins or proteins with altered translational processing will be seen as foreign by the immune system. Antigens that are upregulated or overexpressed (so called self- ns) will not necessarily induce a functional immune response against the tumor: T-cells expressing TCRs that are highly ve to these antigens will have been negatively selected within the thymus in a process known as central nce, meaning that only T-cells with low-affinity TCRs for self-antigens . Therefore, affinity of TCRs or variants of the present description to pepides can be enhanced by methods well known in the art.

The present description further relates to a method of identifying and isolating a TCR according to the present description, said method comprising incubating PBMCs from HLA-A*02-negative healthy donors with A2/peptide monomers, incubating the PBMCs with tetramer—phycoerythrin (PE) and isolating the high avidity T-cells by fluo-rescence ted cell sorting (FACS)—Calibur analysis.

The t description further relates to a method of identifying and isolating a TCR according to the present description, said method comprising ing a transgenic mouse with the entire human TCRdB gene loci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency, immunizing the mouse with a peptide, ting PBMCs obtained from the transgenic mice with er-phycoerythrin (PE), and isolating the high avidity T-cells by fluorescence activated cell sorting (FACS)—Calibur analysis.

In one aspect, to obtain T-cells sing TCRs of the present description, nucleic acids encoding TCR-alpha and/or TCR-beta chains of the present description are cloned into expression vectors, such as gamma retrovirus or lentivirus. The recombinant viruses are generated and then tested for functionality, such as antigen specificity and functional y. An aliquot of the final t is then used to transduce the target T- cell population (generally ed from patient PBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the present description, TCR RNAs are synthesized by techniques known in the art, e.g., in vitro transcription sys- tems. The in vitro-synthesized TCR RNAs are then introduced into primary CD8+ s obtained from y donors by electroporation to re-express tumor specific TCR-alpha and/or ta chains.

To increase the expression, nucleic acids encoding TCRs of the present description may be operably linked to strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate _ 62 _ kinase (PGK), B-actin, ubiquitin, and a simian virus 40 /CD43 composite promoter, elongation factor (EF)—1a and the spleen focus-forming virus (SFFV) promoter. In a preferred embodiment, the er is heterologous to the nucleic acid being expressed.

In addition to strong promoters, TCR expression tes of the present ption may contain additional elements that can enhance transgene expression, including a central polypurine tract (cPPT), which promotes the nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and the woodchuck hepatitis virus posttranscriptional regulatory t (wPRE), which increases the level of transgene expression by increasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may be encoded by nucleic acids located in separate vectors, or may be encoded by polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both the TCR-alpha and TCR-beta chains of the uced TCR be ribed at high levels. To do so, the TCR-alpha and TCR-beta chains of the present description may be cloned into bicistronic constructs in a single vector, which has been shown to be capable of over- coming this obstacle. The use of a viral intraribosomal entry site (IRES) n the TCR-alpha and TCR-beta chains results in the coordinated expression of both chains, because the pha and TCR-beta chains are generated from a single transcript that is broken into two proteins during translation, ensuring that an equal molar ratio of TCR-alpha and TCR-beta chains are produced (Schmitt et al., 2009). c acids encoding TCRs of the present description may be codon zed to increase expression from a host cell. Redundancy in the genetic code allows some amino acids to be encoded by more than one codon, but certain codons are less "op- timal" than others because of the relative bility of matching tRNAs as well as other factors (Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta gene sequences such that each amino acid is encoded by the optimal codon for mammalian gene sion, as well as ating mRNA instability motifs or cryptic splice sites, has been shown to significantly enhance TCR-alpha and TCR-beta gene sion (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chains may result in the acquisition of icities that pose a significant risk for autoimmunity. For e, the formation of mixed TCR dimers may reduce the number of CD3 molecules available to form properly paired TCR complexes, and therefore can significantly decrease the functional avidity of the cells expressing the introduced TCR (Kuball et al., 2007).

To reduce mispairing, the C-terminus domain of the uced TCR chains of the present description may be modified in order to promote interchain affinity, while de- creasing the ability of the introduced chains to pair with the endogenous TCR. These strategies may include replacing the human TCR-alpha and TCR-beta C-terminus domains with their murine counterparts (murinized C-terminus domain); generating a second hain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both the TCR-alpha and TCR-beta chains of the introduced TCR (cysteine modification); swapping interacting residues in the TCR-alpha and TCR-beta chain C-terminus domains ("knob-in-hole"); and fusing the le domains of the TCR-alpha and TCR-beta chains directly to CD3C (CD3C fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of the present description. In preferred embodiments, the host cell is a human T-cell or T-cell progenitor. In some embodiments the T-cell or T-cell progenitor is obtained from a cancer patient. In other embodiments the T-cell or T-cell progenitor is obtained from a healthy donor. Host cells of the present description can be allogeneic or autologous with t to a patient to be treated. In one embodiment, the host is a gamma/delta T-cell transformed to s an alpha/beta TCR.

WO 74645 _ 64 _ A "pharmaceutical composition" is a composition suitable for stration to a human being in a medical setting. Preferably, a pharmaceutical ition is sterile and produced according to GMP guidelines.

The pharmaceutical compositions comprise the peptides or TCR proteins either in the free form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, "a pharmaceutically acceptable salt" refers to a derivative of the disclosed peptides wherein the peptide is modified by making acid or base salts of the agent. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral —NH2 group) involving reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, c acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, c acid, cinnamic acid, ic acid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and the like. Conversely, preparation of basic salts of acid moieties which may be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like. r ment of the present invention relates to a non-naturally occurring peptide wherein said peptide consists or ts essentially of an amino acid sequence ing to SEQ ID No: 1 to SEQ ID No: 188 and has been synthetically produced (e.g. synthesized) as a ceutically acceptable salt. Methods to synthetically produce peptides are well known in the art. The salts of the peptides according to the present invention differ substantially from the peptides in their state(s) in vivo, as the peptides as generated in vivo are no salts. The non-natural salt form of the e mediates the solubility of the peptide, in ular in the context of pharmaceutical compositions comprising the peptides, e.g. the peptide vaccines as disclosed herein. A sufficient and at least substantial solubility of the e(s) is required in order to efficiently provide the peptides to the subject to be treated. Preferably, the salts are pharmaceutically acceptable salts of the peptides. These salts according to the invention include alkaline and earth alkaline salts such as salts of the Hofmeister series comprising as anions P043; 3042', Cchoo; cr, Br', Nog', CIO4', r, SCN' and as cations NH4+, Rb", K", Na", Cs+, Li+, Zn2+, Mg", Ca", Mn", Cu2+ and Ba". Particularly salts are selected from (NH4)3PO4, (NH4)2HPO4, (NH4)H2PO4, (NH4)2SO4, NH4CHgCOO, NH4CI, NH4Br, NH4N03, NH4CIO4, NH4I, NH4SCN, Rb3PO4, szHPO4, RbH2P04, RngO4, Rb4CH3COO, Rb4Cl, Rb4Br, Rb4N03, 4, Rb4l, Rb4SCN, K3PO4, , KH2PO4, K2804, KCchOO, KCI, KBr, KNO3, KCIO4, Kl, KSCN, Na3PO4, Na2HPO4, NaH2PO4, Na2804, NaCHgCOO, NaCl, NaBr, NaN03, NaClO4, Nal, NaSCN, ZhCIz , Cs2HP04, CsH2P04, Cs2804, CSCH3COO, CsCl, CsBr, CsN03, CsClO4, Csl, CsSCN, Li3PO4, Li2HPO4, LiH2PO4, Li2804, OO, LiCl, LiBr, LiNOg, LiClO4, Lil, LiSCN, Cu2804, 4)2, MngPO4, Mg(H2PO4)2, M92804, Mg(CHgCOO)2, MgClg, MgBrg, Mg(N03)2, Mg(ClO4)2, Mglz, Mg(SCN)2, MnClg, Ca3(PO4),, Ca2HP04, Ca(H2PO4)2, CaSO4, Ca(CHgCOO)2, CBCIz, CaBrg, Ca(N03)2, Ca(ClO4)2, Calg, Ca(SCN)2, Ba3(PO4)2, BagHPO4, Ba(H2PO4)2, BaSO4, Ba(CHgCOO)2, BaClg, BaBrg, Ba(N03)2, Ba(ClO4)2, Balg, and Ba(SCN)2. Particularly preferred are NH acetate, MgClg, KH2PO4, Na2804, KCI, NaCl, and CBCIz, such as, for example, the chloride or acetate (trifluoroacetate) salts.

In an especially preferred embodiment, the pharmaceutical compositions comprise the peptides or TCR proteins as salts of acetic acid (acetates), trifluoro es or hloric acid (chlorides).

Preferably, the ment of the present invention is an immunotherapeutic such as a vaccine. It may be administered directly into the patient, into the affected organ or systemically i.d., i.m., s.c., i.p. and iv, or applied ex vivo to cells derived from the t or a human cell line which are subsequently administered to the patient, or used in vitro to select a subpopulation of immune cells derived from the patient, which are then re-administered to the patient. If the nucleic acid is administered to cells in vitro, it may be useful for the cells to be transfected so as to co-express immune-stimulating cytokines, such as eukin-2. The peptide may be ntially pure, or combined with an immune-stimulating adjuvant (see below) or used in combination with immune- stimulatory cytokines, or be administered with a suitable delivery system, for example liposomes. The peptide may also be conjugated to a le carrier such as keyhole limpet haemocyanin (KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). The peptide may also be tagged, may be a fusion n, or may be a hybrid molecule. The peptides whose sequence is given in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of help provided by CD4 er cells. Thus, for MHC Class I epitopes that stimulate CD8 T cells the fusion partner or sections of a hybrid molecule suitably provide epitopes which stimulate CD4-positive T cells. CD4- and CD8-stimulating epitopes are well known in the art and include those identified in the present invention.

In one aspect, the vaccine comprises at least one peptide having the amino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 188, and at least one additional peptide, preferably two to 50, more preferably two to 25, even more ably two to 20 and most preferably two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen peptides. The e(s) may be derived from one or more specific TAAs and may bind to MHC class I molecules.

A further aspect of the invention provides a c acid (for example a polynucleotide) encoding a peptide or e variant of the invention. The polynucleotide may be, for example, DNA, cDNA, PNA, RNA or combinations thereof, either single- and/or double- ed, or native or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothioate backbone and it may or may not contain s so long as it codes for the e. Of course, only peptides that contain naturally occurring amino acid es joined by naturally occurring e bonds are encodable by a polynucleotide. A still further aspect of the invention provides an expression vector capable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini. For instance, complementary _ 67 _ homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding n the mentary homopolymeric tails to form recombinant DNA molecules.

Synthetic s containing one or more restriction sites provide an alternative method of g the DNA segment to vectors. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc. New Haven, CN, USA.

A desirable method of modifying the DNA encoding the polypeptide of the ion employs the polymerase chain on as disclosed by Saiki RK, et aI. (Saiki et a|., 1988). This method may be used for introducing the DNA into a suitable vector, for e by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art. If viral s are used, pox- or adenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then be expressed in a suitable host to produce a polypeptide comprising the peptide or variant of the invention. Thus, the DNA encoding the peptide or variant of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained , to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed, for example, in US 4,440,859, 4,530,901, 4,582,800, 063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired. lly, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and ational regulatory control nucleotide sequences recognized by the desired host, although such ls are generally available in the expression vector. The vector is then uced into the host through standard ques. Generally, not all of the hosts will be transformed by the vector.

Therefore, it will be necessary to select for ormed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance.

Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus spec.), plant cells, animal cells and insect cells. Preferably, the system can be mammalian cells such as CHO cells available from the ATCC Cell Biology tion.

A typical mammalian cell vector plasmid for constitutive expression comprises the CMV or SV4O er with a suitable poly A tail and a resistance , such as neomycin.

One e is pSVL available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia.

Useful yeast d vectors are pRS403-406 and pRS413-416 and are generally _ 69 _ available from Stratagene Cloning s, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere ds (chs). CMV promoter-based vectors (for example from Sigma-Aldrich) provide transient or stable sion, cytoplasmic expression or secretion, and N-terminal or C-terminal tagging in various combinations of FLAG, 3xFLAG, c-myc or MAT. These fusion ns allow for detection, cation and analysis of recombinant protein. Dual-tagged fusions provide flexibility in detection.

The strong human galovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg/L in COS cells. For less potent cell lines, protein levels are lly ~0.1 mg/L. The presence of the SV4O replication origin will result in high levels of DNA replication in SV4O replication permissive COS cells. CMV vectors, for example, can n the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the b-lactamase gene for ampicillin resistance selection in bacteria, hGH polyA, and the f1 origin. Vectors containing the pre-pro-trypsin leader (PPT) ce can direct the secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG antibodies, resins, and plates. Other vectors and expression systems are well known in the art for use with a variety of host cells.

In another embodiment two or more peptides or peptide ts of the invention are encoded and thus expressed in a sive order (similar to "beads on a string" constructs). In doing so, the peptides or peptide variants may be linked or fused together by stretches of linker amino acids, such as for example LLLLLL, or may be linked without any additional peptide(s) between them. These constructs can also be used for cancer therapy, and may induce immune responses both involving MHC l and MHC II.

The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells may be preferred prokaryotic host cells in some _ 70 _ circumstances and lly are a strain of E. coli such as, for e, the E. coli strains DH5 available from Bethesda Research Laboratories Inc, Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and colon cell lines. Yeast host cells include YPH499, YPH500 and YPH501, which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese r ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293 cells which are human embryonic kidney cells.

Preferred insect cells are Sf9 cells which can be ected with baculovirus expression vectors. An overview regarding the choice of suitable host cells for expression can be found in, for example, the textbook of Paulina Balbas and Argelia Lorence "Methods in Molecular Biology Recombinant Gene Expression, s and Protocols," Part One, Second Edition, ISBN 978588299, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well-known methods that typically depend on the type of vector used.

With regard to transformation of yotic host cells, see, for example, Cohen et al.

(Cohen et al., 1972) and (Green and Sambrook, 2012) . Transformation of yeast cells is described in Sherman et al. (Sherman et al., 1986) . The method of Beggs (Beggs, 1978) is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or me formulations, are available from Stratagene Cloning Systems, or Life logies Inc, Gaithersburg, MD 20877, USA. Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells. sfully transformed cells, i.e. cells that contain a DNA construct of the present ion, can be identified by well-known ques such as PCR. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention are useful in the preparation of the peptides of the invention, for example bacterial, yeast and insect cells. However, other host cells may be useful in certain therapeutic methods. For example, antigenpresenting cells, such as dendritic cells, may usefully be used to s the peptides of the invention such that they may be loaded into appropriate MHC molecules. Thus, the current invention provides a host cell comprising a nucleic acid or an expression vector according to the invention.

In a preferred embodiment the host cell is an antigen presenting cell, in particular a dendritic cell or antigen presenting cell. APCs loaded with a recombinant fusion protein containing prostatic acid phosphatase (PAP) were approved by the U.S. Food and Drug Administration (FDA) on April 29, 2010, to treat asymptomatic or minimally symptomatic metastatic HRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing a peptide or its variant, the method comprising culturing a host cell and ing the peptide from the host cell or its culture .

In another embodiment the peptide, the nucleic acid or the expression vector of the invention are used in medicine. For example, the peptide or its variant may be prepared for intravenous (i.v.) injection, sub-cutaneous (s.c.) ion, ermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. Preferred methods of DNA injection include i.d., i.m., s.c., i.p. and iv. Doses of e.g. between 50 pg and 1.5 mg, preferably 125 pg to 500 pg, of peptide or DNA may be given and will depend on the respective peptide or DNA. Dosages of this range were successfully used in previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantially pure, or contained in a suitable vector or delivery system. The nucleic acid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods for designing and introducing such a nucleic acid are well known in the art. An oven/iew is provided by e.g. Teufel et al. (Teufel et al., 2005).

Polynucleotide vaccines are easy to prepare, but the mode of action of these vectors in inducing an immune response is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems based on adenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associated virus or hybrids containing elements of more than one virus. Non-viral delivery s include cationic lipids and cationic polymers and are well known in the art of DNA delivery. Physical ry, such as via a "gene-gun" may also be used. The e or peptides encoded by the c acid may be a fusion n, for example with an epitope that ates T cells for the respective opposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants. Adjuvants are substances that ecifically enhance or potentiate the immune response (e.g., immune responses ed by CD8—positive T cells and helper-T (TH) cells to an antigen, and would thus be considered useful in the medicament of the t invention. Suitable adjuvants e, but are not limited to, 1018 lSS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLlM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, |C30, |C31, lmiquimod (ALDARA®), imod, lmuFact IMP321, Interleukins as lL-2, lL-13, lL-21, Interferon- alpha or -beta, or pegylated derivatives thereof, IS Patch, lSS, ISCOMATRIX, lSCOMs, Juvlmmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide lSA 206, Montanide lSA 50V, Montanide ISA-51, in-oil and oil-in-water ons, OK-432, OM-174, OMMP-EC, ONTAK, OspA, PepTel® vector system, poly(lactid co-glycolid) [PLG]—based and dextran microparticles, talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which is derived from saponin, mycobacterial extracts and synthetic ial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Allison and Krummel, 1995). Also cytokines may be used. l cytokines have been directly linked to influencing tic cell migration to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, lL-1 and lL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its ty) and acting as immunoadjuvants (e.g., lL-12, lL-15, lL-23, lL-7, lFN-alpha. lFN-beta) (Gabrilovich et al., 1996).

CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate daptive) immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigen- ic humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, gous cellular vaccines and polysaccharide conjugates in both prophylactic and therapeutic vaccines.

More importantly it enhances dendritic cell tion and differentiation, resulting in enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias d by TLR9 stimulation is maintained even in the ce of vaccine adjuvants such as alum or incomplete Freund’s adjuvant (IFA) that normally e a TH2 bias. CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar ations, which are especially necessary for inducing a strong response when the antigen is vely weak. They also accelerate the immune response and enable the antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose e without CpG in some experiments (Krieg, 2006). US 6,406,705 Bi describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen- specific immune response. A CpG TLR9 antagonist is dSLlM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany) which is a preferred component of the pharmaceutical composition of the t invention. Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited to chemically modified Cst (e.g. CpR, ldera), dsRNA analogues such as :C) and derivates thereof (e.g.

AmpliGen®, ol®, poly-(ICLC), poly(|C-R), poly(l:C12U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, , AZD2171, anti-CTLA4, other dies ing key structures of the immune system (e.g. anti-CD40, GFbeta, anti-TNFalpha receptor) and 8058175, which may act therapeutically and/or as an nt. The amounts and concentrations of adjuvants and additives useful in the context of the present ion can readily be ined by the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and derivates, |:C) and derivates, RNA, sildenafil, and ulate formulations with PLG or mes.

In a preferred embodiment, the pharmaceutical composition according to the invention the adjuvant is selected from the group consisting of colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according to the invention the adjuvant is selected from the group consisting of colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resiquimod. In a preferred ment of the pharmaceutical composition according to the invention, the adjuvant is _ 75 _ cyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide , poly- ICLC (Hiltonol®) and anti-CD40 mAB, or combinations thereof.

This composition is used for parenteral stration, such as subcutaneous, intradermal, intramuscular or oral administration. For this, the es and optionally other molecules are dissolved or suspended in a pharmaceutically able, preferably aqueous carrier. In addition, the composition can contain excipients, such as buffers, binding agents, ng agents, diluents, s, lubricants, etc. The peptides can also be administered together with immune stimulating substances, such as nes. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for a prevention, prophylaxis and/or therapy of atous or cancerous diseases. Exemplary formulations can be found in, for example, EP2112253.

It is important to realize that the immune response triggered by the vaccine according to the invention attacks the cancer in ent cell-stages and different stages of development. Furthermore ent cancer associated signaling pathways are attacked.

This is an advantage over vaccines that address only one or few targets, which may cause the tumor to easily adapt to the attack (tumor escape). Furthermore, not all individual tumors express the same pattern of antigens. Therefore, a combination of several tumor-associated peptides ensures that every single tumor bears at least some of the targets. The composition is designed in such a way that each tumor is ed to s several of the ns and cover several independent pathways necessary for tumor growth and maintenance. Thus, the vaccine can easily be used "off-the—shelf" for a larger patient population. This means that a pre-selection of patients to be treated with the vaccine can be restricted to HLA typing, does not require any additional biomarker assessments for antigen sion, but it is still ensured that several targets are simultaneously attacked by the induced immune response, which is important for efficacy (Banchereau et al., 2001; Walter et al., 2012).

As used herein, the term "scaffold" refers to a molecule that ically binds to an (e.g. antigenic) determinant. In one embodiment, a scaffold is able to direct the entity to which it is attached (e.g. a (second) antigen binding moiety) to a target site, for example to a ic type of tumor cell or tumor stroma bearing the antigenic determinant (e.g. the complex of a peptide with MHC, according to the application at hand). In another embodiment a scaffold is able to activate ing through its target antigen, for example a T cell receptor complex antigen. Scaffolds include but are not limited to dies and fragments thereof, antigen binding domains of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable , binding proteins comprising at least one ankyrin repeat motif and single domain antigen binding (SDAB) molecules, aptamers, le) TCRs and (modified) cells such as allogenic or autologous T cells. To assess whether a le is a scaffold binding to a target, g assays can be performed.

"Specific" binding means that the scaffold binds the e-MHC-complex of interest better than other naturally occurring peptide-MHC-complexes, to an extent that a scaffold armed with an active molecule that is able to kill a cell bearing the specific target is not able to kill another cell without the specific target but presenting other e-MHC complex(es). Binding to other peptide-MHC complexes is irrelevant if the peptide of the cross-reactive peptide-MHC is not naturally ing, i.e. not derived from the human HLA-peptidome. Tests to assess target cell killing are well known in the art. They should be performed using target cells ry cells or cell lines) with unaltered peptide-MHC presentation, or cells loaded with peptides such that naturally occurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the bound scaffold can be detected by determining the presence or absence of a signal provided by the label. For example, the scaffold can be labelled with a fluorescent dye or any other applicable ar marker molecule. Such marker molecules are well known in the art. For example a fluorescence-labelling, for example provided by a fluorescence dye, can provide a visualization of the bound r by fluorescence or laser scanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such as for example IL- 21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example the background section The present invention further relates to rs. Aptamers (see for example WO 91359 and the literature as cited therein) are short single-stranded nucleic acid les, which can fold into defined three-dimensional structures and recognize specific target structures. They have appeared to be suitable alternatives for ping targeted therapies. rs have been shown to selectively bind to a y of complex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identified within the past decade and provide means for developing diagnostic and eutic approaches.

Since aptamers have been shown to possess almost no toxicity and immunogenicity they are ing candidates for biomedical applications. lndeed aptamers, for example prostate-specific membrane-antigen recognizing aptamers, have been successfully employed for targeted therapies and shown to be functional in xenograft in vivo models. Furthermore, aptamers recognizing specific tumor cell lines have been idenfified.

DNA aptamers can be selected to reveal broad-spectrum recognition properties for various cancer cells, and particularly those derived from solid tumors, while non- tumorigenic and primary healthy cells are not recognized. If the identified rs recognize not only a ic tumor sub-type but rather interact with a series of tumors, this renders the aptamers applicable as so-called broad-spectrum diagnostics and therapeutics.

Further, investigation of cell-binding behavior with flow cytometry showed that the rs revealed very good apparent ties that are within the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, it could be shown that some of the aptamers are taken up by tumor cells and thus can function as molecular vehicles for the targeted delivery of anti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex s such as cells and tissues and complexes of the peptides comprising, preferably consisting of, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 188, according to the invention at hand with the MHC molecule, using the cell-SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique.

The peptides of the present invention can be used to generate and develop specific dies t MHC/peptide complexes. These can be used for therapy, targeting toxins or radioactive substances to the diseased tissue. Another use of these antibodies can be targeting radionuclides to the diseased tissue for imaging purposes such as PET. This use can help to detect small metastases or to determine the size and precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a method for producing a recombinant dy ically binding to a human major histocompatibility complex (MHC) class I or II being complexed with a HLA-restricted antigen, the method comprising: immunizing a genetically engineered non-human mammal comprising cells expressing said human major histocompatibility x (MHC) class I or II with a e form of a MHC class I or II molecule being complexed with said stricted antigen; isolating mRNA molecules from dy producing cells of said non-human mammal; producing a phage y library displaying protein molecules encoded by said mRNA molecules; and isolating at least one phage from said phage display library, _ 7g _ said at least one phage displaying said antibody specifically g to said human major histocompatibility complex (MHC) class I or II being complexed with said HLA- restricted antigen.

It is a further aspect of the invention to provide an antibody that specifically binds to a human major ompatibility complex (MHC) class I or II being complexed with a HLA-restricted antigen, wherein the antibody preferably is a polyclonal antibody, monoclonal antibody, bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain class I major histocompatibility complexes, as well as other tools for the production of these antibodies are disclosed in 03/070752, and in ations (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for the purposes of the present invention are all explicitly incorporated by reference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20 nanomolar, preferably of below 10 nanomolar, to the x, which is also regarded as fic" in the context of the present invention.

The t invention relates to a peptide comprising a sequence that is ed from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 188, or a variant thereof which is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 188 or a variant f that induces T cells reacting with said e, wherein said peptide is not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequence that is selected from the group ting of SEQ ID NO: 1 to SEQ ID NO: 188 or a variant thereof which is at least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 188, wherein said peptide or variant has an overall length of between 8 and 100, preferably between 8 and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to the invention that have the ability to bind to a molecule of the human major histocompatibility complex (MHC) I or -II.

The present invention further relates to the peptides according to the invention wherein the peptide consists or consists essentially of an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 188.

The present invention further relates to the peptides according to the ion, n the peptide is (chemically) ed and/or includes non-peptide bonds.

The present invention further relates to the peptides ing to the invention, wherein the peptide is part of a fusion protein, in particular comprising N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii), or wherein the peptide is fused to (or into) an antibody, such as, for example, an antibody that is specific for dendritic cells.

The t invention further s to a nucleic acid, encoding the peptides according to the invention, provided that the peptide is not the complete (full) human n.

The present invention further relates to the nucleic acid according to the invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable of sing a nucleic acid according to the present invention.

The present invention further relates to a peptide ing to the present invention, a c acid according to the present invention or an expression vector according to the present invention for use in medicine, in particular in the treatment of AML. _ 81 _ The present invention further relates to a host cell comprising a nucleic acid according to the invention or an expression vector according to the invention.

The present invention further relates to the host cell according to the present ion that is an antigen presenting cell, and preferably a dendritic cell.

The present invention further relates to a method of producing a peptide according to the present invention, said method comprising culturing the host cell according to the present invention, and isolating the peptide from said host cell or its culture medium.

The present ion further relates to the method according to the present invention, where-in the n is loaded onto class I or II MHC molecules expressed on the e of a suitable antigen-presenting cell by contacting a sufficient amount of the antigen with an antigen-presenting cell.

The present invention further relates to the method according to the invention, wherein the antigen-presenting cell comprises an expression vector e of expressing said peptide containing SEQ ID NO: 1 to SEQ ID NO: 188 or said t amino acid sequence.

The present invention further relates to activated T cells, produced by the method according to the present invention, wherein said T cells ively recognizes a cell which aberrantly expresses a polypeptide comprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing target cells in a patient which target cells aberrantly s a ptide comprising any amino acid sequence according to the present ion, the method comprising administering to the patient an effective number ofT cells as according to the present invention.

The present invention further relates to the use of any e described, a nucleic acid according to the present invention, an expression vector according to the present invention, a cell according to the present invention, or an activated cytotoxic T lymphocyte according to the present invention as a medicament or in the manufacture of a medicament. The present ion r relates to a use ing to the present invention, wherein the medicament is active against cancer.

The present invention further s to a use according to the invention, wherein the medicament is a vaccine. The present ion r relates to a use according to the invention, wherein the medicament is active against cancer.

The present invention further relates to a use according to the invention, wherein said cancer cells are AML cells or other solid or hematological tumor cells such as bile duct cancer, brain , breast , chronic lymphocytic leukemia, colon or rectum cancer, esophageal cancer, gallbladder cancer, liver cancer, melanoma, dgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney , small cell lung cancer, urinary bladder cancer, uterine cancer.

The present invention further relates to particular marker proteins and biomarkers based on the peptides according to the present invention, herein called "targets" that can be used in the sis and/or prognosis of AML. The present invention also relates to the use of these novel targets for cancer treatment.

The term "antibody" or "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact or "full" immunoglobulin molecules, also included in the term odies" are fragments (e.g. CDRs, Fv, Fab and Fc fragments) or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, as long as they exhibit any of the desired properties (e.g., specific binding of a AML marker (poly)peptide, ry of a toxin to a AML cell expressing a cancer marker gene at an increased level, and/or inhibiting the activity of a AML marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased from commercial sources. The dies of the invention may also be generated using well-known methods. The skilled artisan will understand that either full length AML marker polypeptides or fragments f may be used to generate the dies of the invention. A polypeptide to be used for generating an antibody of the invention may be partially or fully purified from a natural source, or may be produced using recombinant DNA techniques.

For e, a cDNA encoding a e according to the present invention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 188 ptide, or a variant or fragment thereof, can be expressed in prokaryotic cells (e.g., bacteria) or otic cells (e.g., yeast, insect, or ian cells), after which the recombinant protein can be purified and used to generate a monoclonal or polyclonal antibody preparation that specifically bind the AML marker polypeptide used to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or more different sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining an dy with the specificity and affinity ed for its intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). The antibodies are tested for their desired activity by known methods, in accordance with the purpose for which the antibodies are to be used (e.g., ELISA, immunohistochemistry, immunotherapy, etc.; for further guidance on the generation and testing of antibodies, see, e.g., Greenfield, 2014 field, 2014)). For example, the antibodies may be tested in ELISA assays or, Western blots, immunohistochemical staining of formalin- fixed cancers or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods. _ 84 _ The term "monoclonal antibody" as used herein refers to an antibody ed from a substantially homogeneous population of antibodies, i.e.; the individual antibodies comprising the population are identical except for possible lly occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a ular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such dies, so long as they exhibit the desired antagonistic activity (US 4,816,567, which is hereby incorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridoma methods. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit cytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in US 567. DNA encoding the onal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

In vitro methods are also le for preparing lent antibodies. Digestion of antibodies to e fragments thereof, particularly Fab fragments, can be accomplished using e techniques known in the art. For instance, digestion can be performed using papain. es of papain digestion are described in WO 94/29348 and US 4,342,566. Papain ion of antibodies typically produces two identical antigen g fragments, called Fab fragments, each with a single antigen binding _ 85 _ site, and a residual Fc fragment. Pepsin treatment yields a F(ab')2 fragment and a ch' fragment.

The antibody fragments, whether attached to other sequences or not, can also include ions, deletions, substitutions, or other selected modifications of ular regions or specific amino acids residues, ed the activity of the fragment is not significantly altered or impaired compared to the dified antibody or antibody fragment. These cations can provide for some additional property, such as to remove/add amino acids e of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc.

Functional or active regions of the dy may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanized antibodies or human dies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, globulin chains or fragments thereof (such as Fv, Fab, Fab' or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. zed antibodies include human immunoglobulins (recipient antibody) in which residues from a mentary determining region (CDR) of the recipient are replaced by es from a CDR of a non-human species (donor dy) such as mouse, rat or rabbit having the d specificity, affinity and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.

Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In l, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as t" residues, which are lly taken from an "import" variable domain.

Humanization can be essentially med by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (US 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In ce, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent dies.

Transgenic s (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous on of the antibody heavy chain joining region gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. er of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon n challenge. Human dies can also be produced in phage display libraries. dies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation ic. Examples of the pharmaceutically-acceptable carrier include saline, Ringer's on and dextrose solution. The pH of the solution is preferably from about 5 to _ 87 _ about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release ations such as semipermeable es of solid hydrophobic polymers containing the antibody, which es are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for ce, the route of administration and concentration of antibody being administered.

The antibodies can be administered to the t, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. The antibodies may also be administered by intratumoral or peritumoral routes, to exert local as well as systemic therapeutic effects. Local or intravenous injection is preferred.

Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be stered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the ular type of antibody used and other drugs being administered.

A typical daily dosage of the antibody used alone might range from about 1 (pg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. Following administration of an antibody, ably for treating AML, the efficacy of the therapeutic antibody can be assessed in various ways well known to the skilled tioner. For instance, the size, number, and/or distribution of cancer in a subject receiving treatment may be monitored using standard tumor imaging techniques. A therapeutically-administered antibody that arrests tumor growth, results in tumor shrinkage, and/or ts the development of new tumors, compared to the disease course that would occurs in the absence of antibody administration, is an efficacious antibody for ent of cancer.

It is a further aspect of the invention to provide a method for producing a soluble T-cell receptor (sTCR) recognizing a specific peptide-MHC complex. Such soluble T-cell _ 88 _ ors can be generated from specific T-cell clones, and their affinity can be increased by mutagenesis targeting the complementarity-determining regions. For the purpose of T-cell receptor selection, phage display can be used (US 2010/0113300, (Liddy et al., 2012)). For the purpose of ization of T-cell receptors during phage display and in case of practical use as drug, alpha and beta chain can be linked e.g. by non-native disulfide bonds, other covalent bonds (single-chain T-cell receptor), or by dimerization domains (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999). The T-cell receptor can be linked to toxins, drugs, nes (see, for example, US 2013/0115191), and domains recruiting effector cells such as an anti-CD3 domain, etc., in order to execute particular functions on target cells. Moreover, it could be expressed in T cells used for ve transfer. Further information can be found in WC 2004/033685A1 and 56407A1. r methods for the production are disclosed in WC 57586A1.

In addition, the peptides and/or the TCRs or dies or other binding molecules of the present invention can be used to verify a pathologist’s diagnosis of a cancer based on a biopsied .

The antibodies or TCRs may also be used for in vivo diagnostic assays. Generally, the antibody is labeled with a radionucleotide (such as 111in, 99To, 14c, 131i, 3H, 32p or 353) so that the tumor can be localized using immunoscintiography. In one embodiment, antibodies or fragments thereof bind to the extracellular domains of two or more targets of a protein selected from the group consisting of the above-mentioned proteins, and the affinity value (Kd) is less than 1 x 10pM.

Antibodies for diagnostic use may be labeled with probes suitable for detection by s imaging methods. Methods for detection of probes include, but are not limited to, fluorescence, light, al and on microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography and positron emission tomography. Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine-18 and other positron-emitting radionuclides. Additionally, probes may be bi- or multi-functional and be detectable by more than one of the s listed. These antibodies may be directly or indirectly labeled with said probes.

Attachment of probes to the antibodies includes covalent attachment of the probe, oration of the probe into the antibody, and the covalent attachment of a chelating compound for binding of probe, amongst others well recognized in the art. For immunohistochemistry, the disease tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as in. The fixed or embedded section contains the sample are contacted with a labeled y antibody and secondary antibody, wherein the dy is used to detect the expression of the proteins in situ. r aspect of the present invention includes an in vitro method for ing activated T cells, the method comprising contacting in vitro T cells with antigen loaded human MHC molecules expressed on the surface of a suitable n-presenting cell for a period of time sufficient to activate the T cell in an antigen specific manner, n the antigen is a e according to the invention. Preferably a sufficient amount of the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or function of the TAP peptide transporter. Suitable cells that lack the TAP peptide transporter e T2, RMA-S and Drosophila cells. TAP is the transporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA under Catalogue No CRL 1992; the Drosophila cell line Schneider line 2 is available from the ATCC under Catalogue No CRL 19863; the mouse RMA-S cell line is described in Ljunggren et al. (Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially no MHC class I molecules. It is also preferred that the ator cell expresses a molecule important for providing a co-stimulatory signal for T-cells such as any of 87.1, 87.2, lCAM-1 and LFA 3. The nucleic acid sequences of numerous MHC class I molecules and of the co- stimulator molecules are publicly available from the GenBank and EMBL ses.

In case of a MHC class I e being used as an antigen, the T cells are CD8—positive T cells.

If an antigen-presenting cell is transfected to express such an epitope, preferably the cell comprises an expression vector capable of sing a e containing SEQ ID NO: 1 to SEQ ID NO: 188, or a variant amino acid ce thereof.

A number of other methods may be used for generating T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used in the generation of CTL.

Plebanski et al. (Plebanski et al., 1995) made use of gous peripheral blood lymphocytes (PLBs) in the preparation of T cells. Furthermore, the tion of autologous T cells by pulsing dendritic cells with peptide or polypeptide, or via infection with recombinant virus is possible. Also, B cells can be used in the production of gous T cells. In addition, macrophages pulsed with peptide or polypeptide, or infected with recombinant virus, may be used in the preparation of autologous T cells. 8. Walter et al. (Walter et al., 2003) describe the in vitro priming of T cells by using cial antigen presenting cells (aAPCs), which is also a suitable way for generating T cells t the peptide of choice. In the present invention, aAPCs were generated by the coupling of preformed MHC:peptide complexes to the surface of polystyrene particles (microbeads) by biotin:streptavidin biochemistry. This system permits the exact control of the MHC density on aAPCs, which allows to selectively eliciting high- or low- avidity antigen-specific T cell responses with high efficiency from blood samples. Apart from MHC:peptide complexes, aAPCs should carry other proteins with co-stimulatory activity like anti-CD28 antibodies coupled to their surface. Furthermore such aAPC- based systems often require the addition of appropriate soluble s, e. g. cytokines, like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and a method is described in detail in WO 28, incorporated herein by reference. For example, in addition to Drosophila cells and T2 cells, other cells may be used to present antigens such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and ia- infected target cells. In addition plant viruses may be used (see, for example, Porta et al. (Porta et al., 1994) which describes the development of cowpea mosaic virus as a high-yielding system for the presentation of n peptides.

The activated T cells that are directed against the peptides of the invention are useful in therapy. Thus, a further aspect of the ion es activated T cells obtainable by the foregoing methods of the invention.

Activated T cells, which are produced by the above method, will selectively recognize a cell that aberrantly expresses a polypeptide that comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 188.

Preferably, the T cell recognizes the cell by interacting through its TCR with the HLA/peptide-complex (for example, binding). The T cells are useful in a method of killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention wherein the t is administered an effective number of the activated T cells. The T cells that are administered to the patient may be derived from the patient and activated as described above (i.e. they are autologous T . Alternatively, the T cells are not from the patient but are from another individual. Of course, it is preferred if the dual is a healthy individual. By "healthy dual" the inventors mean that the individual is generally in good health, preferably has a competent immune system and, more preferably, is not suffering from any disease that can be readily tested for, and detected. _ 92 _ In vivo, the target cells for the CD8—positive T cells according to the present ion can be cells of the tumor (which sometimes express MHC class II) and/or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class II; el et al., 2006)).

The T cells of the present invention may be used as active ingredients of a therapeutic composition. Thus, the invention also provides a method of killing target cells in a patient whose target cells aberrantly express a polypeptide comprising an amino acid sequence of the invention, the method comprising administering to the patient an effective number of T cells as defined above.

By "aberrantly expressed" the inventors also mean that the polypeptide is over- sed compared to levels of expression in normal tissues or that the gene is silent in the tissue from which the tumor is derived but in the tumor it is expressed. By "over- expressed" the inventors mean that the polypeptide is present at a level at least ld of that present in normal tissue; preferably at least 2—fold, and more ably at least -fold or 10-fold the level present in normal tissue.

T cells may be ed by methods known in the art, e.g. those described above.

Protocols for this so-called adoptive transfer of T cells are well known in the art.

Reviews can be found in: Gattioni et al. and Morgan et al. (Gattinoni et al., 2006; Morgan et al., 2006). r aspect of the present invention includes the use of the es complexed with MHC to generate a T-cell receptor whose nucleic acid is cloned and is introduced into a host cell, preferably a T cell. This engineered T cell can then be transferred to a patient for therapy of cancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T-cell receptor or the nucleic acid encoding it, is useful for the _ g3 _ ent of disorders, characterized by cells escaping an immune response. ore any molecule of the t invention may be used as medicament or in the manufacture of a medicament. The molecule may be used by itself or combined with other molecule(s) of the invention or (a) known molecule(s).

The t invention is further directed at a kit comprising: (a) a container containing a pharmaceutical composition as described above, in solution or in lyophilized form; (b) optionally a second container containing a diluent or reconstituting solution for the lyophilized formulation; and (c) optionally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a , (vi) a needle, or (v) a syringe. The container is ably a bottle, a vial, a syringe or test tube; and it may be a multi-use container. The pharmaceutical composition is ably lyophmzed.

Kits of the present invention preferably comprise a lyophilized formulation of the present invention in a suitable container and instructions for its reconstitution and/or use.

Suitable containers include, for example, bottles, vials (e.g. dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. Preferably the kit and/or container n/s instructions on or associated with the container that indicates directions for reconstitution and/or use. For example, the label may indicate that the lized formulation is to be reconstituted to peptide concentrations as described above. The label may further indicate that the formulation is useful or intended for subcutaneous administration.

The container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation. The kit _ g4 _ may further comprise a second ner sing a suitable diluent (e.g., sodium bicarbonate solution).

Upon mixing of the diluent and the lyophilized formulation, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg/mL/peptide (=75 pg) and preferably not more than 3 mg/mL/peptide (=1500 pg). The kit may further include other materials desirable from a commercial and user standpoint, including other s, diluents, filters, needles, syringes, and package inserts with instructions for use.

Kits of the present invention may have a single ner that contains the formulation of the pharmaceutical itions according to the present invention with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component. ably, kits of the invention include a formulation of the invention packaged for use in combination with the co-administration of a second compound (such as adjuvants (e.g. GM-CSF), a herapeutic agent, a l product, a hormone or antagonist, an anti-angiogenesis agent or inhibitor, an apoptosis-inducing agent or a chelator) or a ceutical composition thereof. The components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient. The components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution. The components of the kit may also be provided as solids, which may be converted into s by addition of suitable solvents, which are ably provided in another distinct container.

The ner of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid. Usually, when there is more than one component, the kit will contain a second vial or other container, which allows for separate dosing. The kit may also contain another container for a pharmaceutically WO 74645 _ g5 _ acceptable liquid. Preferably, a therapeutic kit will contain an apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the agents of the invention that are components of the present kit.

The present formulation is one that is suitable for administration of the peptides by any able route such as oral al), nasal, ophthal, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, the stration is so, and most preferably i.d. stration may be by infusion pump.

Since the peptides of the invention were isolated from AML, the medicament of the invention is preferably used to treat AML.

The present invention r relates to a method for producing a alized pharmaceutical for an individual patient comprising manufacturing a pharmaceutical composition comprising at least one e selected from a use of prescreened TUMAPs, wherein the at least one peptide used in the pharmaceutical composition is selected for suitability in the individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The method could also be adapted to produce T cell clones for down-stream applications, such as TCR isolations, or e antibodies, and other treatment options.

A "personalized ceutical" shall mean ically tailored therapies for one individual patient that will only be used for therapy in such individual patient, including actively personalized cancer vaccines and adoptive cellular therapies using autologous paflentfissue.

As used herein, the term "warehouse" shall refer to a group or set of peptides that have been pre-screened for immunogenicity and/or over-presentation in a particular tumor type. The term "warehouse" is not intended to imply that the particular peptides included in the vaccine have been pre-manufactured and stored in a physical facility, although that possibility is contemplated. It is expressly contemplated that the peptides may be _ 96 _ manufactured de novo for each dualized vaccine produced, or may be pre- manufactured and stored. The warehouse (e.g. in the form of a database) is composed of associated peptides which were highly overexpressed in the tumor tissue of AML patients with various HLA-A HLA-B and HLA-C alleles. It may n MHC class I and MHC class II peptides or elongated MHC class I es. In addition to the tumor associated peptides collected from several AML tissues, the warehouse may contain HLA-A*02 and HLA-A*24 marker peptides. These peptides allow comparison of the magnitude of T-cell ty induced by TUMAPS in a quantitative manner and hence allow ant conclusion to be drawn on the capacity of the vaccine to elicit anti-tumor responses. Secondly, they function as important ve control peptides derived from a "non-self" antigen in the case that any vaccine-induced T-cell responses to TUMAPs derived from "self" antigens in a patient are not observed. And y, it may allow conclusions to be drawn, regarding the status of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integrated functional genomics approach combining gene expression analysis, mass spectrometry, and T-cell immunology (XPresident ®). The approach assures that only TUMAPs truly present on a high percentage of tumors but not or only minimally expressed on normal tissue, are chosen for r analysis. For initial peptide selection, AML samples from patients and blood from healthy donors were analyzed in a stepwise approach: 1. HLA s from the malignant material were identified by mass spectrometry 2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis was used to identify genes over-expressed in the malignant tissue (AML) compared with a range of normal organs and tissues 3. Identified HLA ligands were compared to gene expression data. Peptides over- presented or selectively presented on tumor tissue, preferably d by selectively expressed or xpressed genes as detected in step 2 were considered suitable TUMAP candidates for a multi-peptide vaccine. 4. Literature research was performed in order to identify additional ce supporting the relevance of the identified peptides as TUMAPs . The relevance of over-expression at the mRNA level was confirmed by redetection of selected TUMAPs from step 3 on tumor tissue and lack of (or infrequent) detection on healthy tissues. 6. In order to assess, whether an induction of in vivo T-cell responses by the selected peptides may be feasible, in vitro immunogenicity assays were med using human T cells from healthy donors as well as from AML patients.

In an aspect, the peptides are pre-screened for immunogenicity before being included in the use. By way of example, and not limitation, the immunogenicity of the es included in the warehouse is ined by a method comprising in vitro T-cell priming through repeated stimulations of CD8+ T cells from healthy donors with artificial antigen presenting cells loaded with peptide/MHC complexes and D28 antibody.

This method is preferred for rare cancers and ts with a rare expression profile. In contrast to multi-peptide cocktails with a fixed composition as currently developed, the warehouse allows a significantly higher matching of the actual expression of antigens in the tumor with the vaccine. ed single or combinations of several "off-the-shelf’ peptides will be used for each patient in a multitarget approach. In theory an approach based on ion of e.g. 5 different antigenic peptides from a library of 50 would already lead to approximately 17 million possible drug product (DP) compositions.

In an aspect, the peptides are selected for ion in the vaccine based on their suitability for the individual patient based on the method according to the t invention as bed herein, or as below.

The HLA phenotype, transcriptomic and omic data is gathered from the patient’s tumor material, and blood samples to identify the most suitable peptides for each patient containing "warehouse" and patient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen, which are selectively or over-expressed in the patients tumor and, where possible, show strong in vitro immunogenicity if tested with the patients’ individual PBMCs.

WO 74645 2017/058083 Preferably, the peptides ed in the vaccine are identified by a method comprising: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the individual patient; (b) comparing the es identified in (a) with a warehouse (database) of peptides as described above; and (c) selecting at least one peptide from the warehouse (database) that correlates with a associated peptide identified in the patient. For example, the TUMAPs presented by the tumor sample are identified by: (a1) comparing expression data from the tumor sample to expression data from a sample of normal tissue corresponding to the tissue type of the tumor sample to identify proteins that are over-expressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with sequences of MHC ligands bound to MHC class I and/or class II molecules in the tumor sample to identify MHC s derived from proteins over-expressed or aberrantly expressed by the tumor. Preferably, the sequences of MHC ligands are identified by eluting bound peptides from MHC molecules isolated from the tumor , and sequencing the eluted ligands. ably, the tumor sample and the normal tissue are obtained from the same patient.

In addition to, or as an alternative to, selecting peptides using a warehousing (database) model, TUMAPs may be identified in the patient de novo, and then included in the vaccine. As one example, candidate TUMAPs may be identified in the patient by (a1) comparing expression data from the tumor sample to expression data from a sample of normal tissue corresponding to the tissue type of the tumor sample to identify proteins that are over-expressed or aberrantly expressed in the tumor sample; and (a2) correlating the expression data with sequences of MHC ligands bound to MHC class I and/or class II molecules in the tumor sample to identify MHC ligands derived from proteins over-expressed or aberrantly sed by the tumor. As another example, proteins may be identified containing mutations that are unique to the tumor sample ve to normal ponding tissue from the individual patient, and TUMAPs can be identified that specifically target the mutation. For example, the genome of the tumor and of corresponding normal tissue can be sequenced by whole genome sequencing: For discovery of non-synonymous mutations in the protein-coding regions of genes, _ gg _ genomic DNA and RNA are extracted from tumor tissues and normal non-mutated c germline DNA is extracted from peripheral blood mononuclear cells (PBMCs).

The applied NGS ch is confined to the re-sequencing of protein coding regions (exome re-sequencing). For this purpose, exonic DNA from human samples is captured using vendor-supplied target enrichment kits, followed by sequencing with e.g. a HiSeq2000 (lllumina). Additionally, tumor mRNA is sequenced for direct quantification of gene expression and validation that mutated genes are expressed in the patients’ tumors. The resultant millions of sequence reads are processed through software algorithms. The output list contains mutations and gene expression. Tumor-specific c mutations are determined by comparison with the PBMC-derived germline variations and tized. The de novo identified peptides can then be tested for immunogenicity as described above for the warehouse, and candidate TUMAPs possessing suitable genicity are ed for ion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the individual patient by the method as described above; (b) comparing the peptides identified in a) with a warehouse of peptides that have been prescreened for immunogenicity and overpresentation in tumors as compared to corresponding normal ; (c) selecting at least one peptide from the warehouse that correlates with a associated peptide identified in the patient; and (d) optionally, selecting at least one peptide identified de novo in (a) confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by a tumor sample from the dual patient; and (b) selecting at least one e identified de novo in (a) and confirming its immunogenicity.

Once the peptides for a personalized peptide based e are selected, the vaccine is produced. The vaccine preferably is a liquid formulation consisting of the individual peptides dissolved in between 20-40% DMSO, preferably about 30-35% DMSO, such as about 33% DMSO.

Each e to be ed into a product is ved in DMSO. The concentration of the single peptide solutions has to be chosen depending on the number of peptides to be included into the product. The single peptide-DMSO solutions are mixed in equal parts to achieve a solution containing all peptides to be included in the product with a concentration of ~2.5 mg/ml per peptide. The mixed solution is then diluted 1:3 with water for injection to achieve a concentration of 0.826 mg/ml per peptide in 33% DMSO.

The diluted solution is filtered through a 0.22 pm e filter. The final bulk on is obtained.

Final bulk solution is filled into vials and stored at -20°C until use. One vial contains 700 pL solution, containing 0.578 mg of each peptide. Of this, 500 pL (approx. 400 pg per peptide) will be applied for intradermal injection.

In addition to being useful for treating cancer, the peptides of the present invention are also useful as diagnostics. Since the peptides were generated from AML cells and since it was determined that these peptides are not or at lower levels t in normal tissues, these es can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples can assist a pathologist in diagnosis of . Detection of certain peptides by means of antibodies, mass spectrometry or other methods known in the art can tell the pathologist that the tissue sample is malignant or inflamed or generally diseased, or can be used as a biomarker for AML. Presence of groups of peptides can enable classification or sub- classification of ed tissues.

The detection of peptides on diseased tissue specimen can enable the decision about the benefit of therapies involving the immune system, especially if T-lymphocytes are known or expected to be involved in the ism of action. Loss of MHC expression is a well described mechanism by which infected of malignant cells escape immuno- sun/eillance. Thus, presence of peptides shows that this mechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyze lymphocyte responses against those es such as T cell responses or antibody ses t the e or the peptide complexed to MHC molecules. These lymphocyte ses can be used as prognostic markers for decision on further therapy steps. These responses can also be used as surrogate response markers in immunotherapy approaches aiming to induce lymphocyte responses by different means, e.g. vaccination of protein, c acids, autologous materials, adoptive transfer of lymphocytes. In gene therapy settings, lymphocyte responses against peptides can be considered in the assessment of side effects. Monitoring of lymphocyte responses might also be a valuable tool for -up examinations of transplantation therapies, e.g. for the detection of graft versus host and host versus graft diseases.

The present ion will now be described in the following examples which describe red embodiments f, and with nce to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their ties.

FIGURES Figures 1A through 1D show the over-presentation of various peptides in normal tissues (white bars) and AML (black bars). Figure 1A), Gene symbol: GNA15, Peptide: LLDSAVYYL (SEQ ID NO.: 1) Tissues from left to right: 5 adipose tissues, 5 adrenal glands, 15 blood vessels, 14 brains, 7 breasts, 7 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 20 large intestines, 24 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 8 pancreases, 6 parathyroid glands, 1 peritoneum, 5 pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 5 skeletal muscles, 11 skins, 4 small intestines, 12 spleens, 5 stomachs, 5 testes, 2 thymi, 2 thyroid , 11 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 24 blood cells, 10 bone marrows, 17 AML samples.

The peptide has additionally been detected on 1/17 gallbladder and bile duct cancers, 2/18 melanomas, 1/20 ovarian cancers, 2/17 geal cancers, 4/85 all cell lung cancers, 1/15 urinary bladder cancers and 1/16 uterine cancers. Figure 1B) Gene symbol: DDX50, Peptide: MEL (SEQ ID NO.: 7) Tissues from left to right: 5 adipose tissues, 5 adrenal glands, 15 blood vessels, 14 brains, 7 breasts, 7 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 20 large ines, 24 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 8 pancreases, 6 parathyroid glands, 1 peritoneum, 5 pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 5 skeletal muscles, 11 skins, 4 small intestines, 12 spleens, 5 stomachs, 5 testes, 2 thymi, 2 thyroid glands, 11 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 24 blood cells, 10 bone marrows, 17 AML s. The peptide has additionally been detected on 2/20 non-hodgkin lymphomas. Figure 10) Gene symbols: TMEM183B,TMEM183A, e: VlLDPVHSV (SEQ ID NO.: 10) Tissues from left to right: 5 adipose s, 5 l glands, 15 blood vessels, 14 brains, 7 breasts, 7 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 20 large intestines, 24 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 8 pancreases, 6 parathyroid glands, 1 peritoneum, 5 pituitary glands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 5 skeletal muscles, 11 skins, 4 small intestines, 12 spleens, 5 stomachs, 5 , 2 thymi, 2 thyroid glands, 11 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 24 blood cells, 10 bone marrows, 17 AML samples.

The peptide has additionally been detected on 1/18 breast cancers, 2/20 non-hodgkin lymphomas, 1/20 ovarian cancers, 1/85 all cell lung cancers, 1/17 small cell lung s, 1/16 uterine cancers. Figure 1 D) Gene symbol: TRIM27, Peptide: RQV (SEQ ID NO.: 138) Samples from left to right: 2 cancer cell lines, 1 primary cancer culture, 5 normal tissues (1 adrenal gland, 1 bone marrow, 1 colon, 1 kidney, 1 stomach), 34 cancer tissues (2 bone marrow cancers, 2 breast cancers, 1 esophageal , 1 kidney cancer, 3 leukocytic leukemia cancers, 2 liver cancers, 8 lung s, lymph node cancers, 2 myeloid cells cancers, 1 ovarian cancer, 1 prostate cancer, 1 rectum cancer, 2 skin cancers, 3 urinary r cancers).

Figures 2A) through 20) show exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in AML in a panel of normal tissues (white bars) and 11 AML samples (black bars). Tissues from left to right: 6 arteries, 2 blood cells, 2 , 1 heart, 2 livers, 3 lungs, 2 veins, 1 e tissue, 1 adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 4 pancreases, 2 peripheral nerves, 2 pituitary glands, 1 rectum, 2 salivary glands, 2 skeletal muscles, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1 thyroid gland, 7 tracheas, 1 urinary bladder, 1 , 5 ovaries, 5 placentas, 1 prostate, 1 , 1 thymus, 1 uterus, 11 AML samples. Figure 2A) Gene symbol: COL24A1, Figure 28) Gene symbol: SPNS3, Figure 2C) Gene symbol: KCNE1L.

Figure 1 shows exemplary immunogenicity data: flow cytometry results after e- specific multimer staining.

EXAMPLES EXAMPLE 1 Identification and guantitation of tumor associated peptides ted on the cell surface Tissue samples Patients’ tumor tissues were obtained from: ProteoGenex lnc. (Culver City, CA, USA) Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn, Germany); University Hospital Ttibingen (Ttibingen, Germany).

Normal tissues were obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK) tions lnc. (Brea, CA, USA); BioServe (Beltsville, MD, USA); Capital BioScience lnc. (Rockville, MD, USA); Geneticist lnc. ale, CA, USA); Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan); Genex lnc. (Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva (Geneva, Switzerland); University Hospital Heidelberg (Heidelberg, Germany); University al Munich (Munich, Germany); University Hospital Ttibingen ngen, Germany). -1o4- Written informed ts of all patients had been given before surgery or autopsy.

Tissues were shock-frozen ately after excision and stored until isolation of TUMAPs at -70°C or below.

Isolation of HLA peptides from tissue samples HLA peptide pools from shock-frozen tissue samples were obtained by immune precipitation from solid tissues according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999) using the HLA-A*02—specific antibody 887.2, the HLA-A, -B, - C-specific antibody WES/32, CNBr—activated sepharose, acid treatment, and ultrafiltration.

Mass spectrometry analyses The HLA peptide pools as obtained were separated according to their hydrophobicity by ed-phase chromatography (nanoAcquity UPLC system, Waters) and the eluting es were analyzed in LTQ- velos and fusion hybrid mass spectrometers oElectron) equipped with an ESI source. Peptide pools were loaded directly onto the analytical fused-silica micro-capillary column (75 pm id x 250 mm) packed with 1.7 pm C18 reversed-phase material s) applying a flow rate of 400 nL per minute.

Subsequently, the peptides were separated using a two-step 180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoESl source. The LTQ-Orbitrap mass spectrometers were operated in the data- dependent mode using a TOP5 strategy. In brief, a scan cycle was initiated with a full scan of high mass accuracy in the orbitrap (R = 30 000), which was followed by MS/MS scans also in the orbitrap (R = 7500) on the 5 most abundant precursor ions with dynamic ion of previously selected ions. Tandem mass spectra were reted by SEQUEST and additional manual l. The identified e sequence was assured by comparison of the ted natural peptide fragmentation pattern with the fragmentation pattern of a synthetic sequence-identical reference peptide.

Label-free relative LC-MS quantitation was performed by ion counting i.e. by extraction and analysis of LC-MS features (Mueller et al., 2007). The method assumes that the peptide’s LC-MS signal area correlates with its abundance in the sample. Extracted features were further processed by charge state deconvolution and retention time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features were cross-referenced with the sequence fication results to combine quantitative data of different samples and tissues to peptide presentation profiles. The quantitative data were normalized in a two-tier fashion according to central tendency to account for ion within technical and biological replicates. Thus each identified peptide can be ated with quantitative data ng relative quantification between samples and tissues. In on, all quantitative data acquired for peptide candidates was inspected manually to assure data consistency and to verify the accuracy of the automated analysis. For each peptide a tation profile was calculated showing the mean sample presentation as well as replicate variations. The es ose AML samples to a baseline of normal tissue samples. Presentation profiles of ary over- presented peptides are shown in Figure 1. Presentation scores for exemplary peptides are shown in Table 8.

Table 8: Presentation scores. The table lists peptides that are very highly over- presented on tumors compared to a panel of normal tissues (+++), highly over- presented on tumors compared to a panel of normal tissues (++) or over-presented on tumors compared to a panel of normal tissues (+).The panel of normal tissues considered relevant for comparison with tumors consisted of: adipose , adrenal gland, , blood cells, bone marrow, brain, central nerve, colon, duodenum, esophagus, eye, adder, heart, kidney, liver, lung, lymph node, as, parathyroid gland, peripheral nerve, peritoneum, pituitary gland, pleura, rectum, ry gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, ureter, urinary bladder, vein.

SEQ ID Peptide Sequence No. Presentation 1 LLDSAVYYL +++ 2 VLLKAVAQA +++ 3 ALYDKTKRIFL +++ 4 FLPDAFVTM +++ FLYYEDLVSC +++ 7 LLWGDIMEL +++ 8 LLWPGAALLV +++ VILDPVHSV +++ 11 ILTQIDHIL +++ 12 ALIESNTAL +++ 13 ALVPGVTQV +++ 14 ALWWGTITL +++ FIDEEVEDMYL +++ 16 FLDTQAPSL +++ 17 FLLGLSEQL +++ 18 GIIEENWQL +++ 19 GIVEYLSLV +++ GLDAFLLEL +++ 21 GLFHGTELL +++ 22 GLLQLDTAFV +++ 23 GLLQPPVRIV +++ 24 NRV +++ GVEGSLIVEKI +++ 26 NAGVEGSLIVEKI +++ 27 KANPALWL +++ 28 LLDQMETPL +++ 29 RLGPSWGL +++ SAL +++ 31 SLFVFIPMV +++ 32 WHL +++ 33 TIMNQEKLAKL +++ VLFEHAVGYAL +++ 36 VLGPSPSSV +++ 37 VVAPAPVVEAV +++ 38 AAIASTPTL +++ 39 AIFAGTMQL +++ 40 ALAAGGYDVEKN +++ 41 ALFILPFVSV +++ 42 ALTTYTIEV +++ 43 AMLDFVSSL +++ 44 FAVDNVGNRTL +++ 45 FLFTDVLLM +++ 46 GLDQYLQEV +++ 47 GLIJPNVQL +++ 48 IAIEALTQL +++ 49 IIDDNHAIV +++ 50 IIWATSLLL +++ 51 SLLSSSLNV +++ 52 IVDPVDSTL +++ 53 KAFLGELTL +++ 54 KLPEFLVQL +++ 55 KTLDLINKL +++ 56 LANPTTSAL +++ 57 LLDFGSLSNLQV +++ 58 LLLATLQEA +++ 59 AIVSL +++ 60 NLLNVLEYL +++ 61 FLLPGVLLSEA +++ 62 RLLFNLSEV +++ 63 RLNDTIQLL +++ 64 SLANIKIWV +++ 65 SLEEQLSALTL +++ 66 SLKNEVGGLV +++ 67 SLQDRVIAL +++ 68 TGITTPVASV +++ 69 TIIGLVRVI +++ 70 AQL +++ 71 TLTSSLATV +++ 72 DASSL +++ 73 VAIPDVDPL +++ 74 VANPVLWL +++ 75 VLAPLGFTL +++ 76 VLLJPVPEL +++ 77 VLNMKPPEI +++ 78 VLSEVECHL +++ 79 YLMDPDTFTF +++ 80 YLTEALQSI +++ 81 YVTEELPQL +++ 82 LLPDNFIAA ++ 84 GLLGSVLTI +++ 85 LYL +++ 86 HLLGDPMANV + 87 ILKPFGNSI +++ 88 LALNFGSTL +++ 89 LLESPVDGWQV +++ 90 LLLDTVTSI +++ 91 RLAHYIDRV +++ 92 RLWDIQHQL +++ 93 SLINDVLAE +++ 94 SLLEFAQYL +++ 95 SVAEINVLI +++ 96 TLLASYVFL +++ 97 TIMTGVIGV ++ 98 TQFGFLMEV +++ 99 YLAPFSLSNY +++ 100 AAPAVLGEVDTSLV +++ 101 AINKDPEAPIFQV + 102 ERV +++ 103 ALGDFGIRL ++ 104 ALIPETTTL ++ 105 GVFALVTAV + 106 ALLEELERSTL + 107 ALLGMLPLL +++ 108 ELEMNSDLKAQL +++ 109 GLLAVPLLAA +++ 110 VVPLDLV +++ 111 GVEPAADGKGWW +++ 112 ILRDALNQA ++ 113 NLQSEVEGV +++ 1 14 RLAQEAAQV ++ 115 SLPDLTTPL +++ 116 TILEILPEL +++ 117 TILPTILFL ++ 1 18 TLLTVLTQA +++ 119 TLTDELAAL +++ 120 VIQDLWSV +++ 121 VLQAGQYGV +++ 122 VLL +++ 123 YTVKINPTL +++ 124 GLPELVIQL +++ 125 GLFGYLVFL ++ 126 IQAV ++ 127 KIISALPQL ++ 128 NLSTKTEAV +++ 129 RMAVLNEQV + 130 GVLGNALEGV + 131 SLFSGSLEPV + 132 SLYPVLNFL ++ 133 TVIGTLLFL + 135 GIIDRIFQA ++ 136 GLSSIETLL +++ 137 ILAPLAWDL +++ 139 NLIIFSPSV + 141 GLLPPLRIPELL ++ 142 GLSDGYGFTT +++ 143 YLLPHILVY +++ 144 GLFMGLVLV + 147 FALPILNAL +++ 148 FLYFEDHGL ++ 149 GLAEILVLV + 151 GLLPFPEVTL + 152 GLSNHIAAL ++ 153 LAL +++ 154 IIADNIIFL ++ 155 ILDLIQVFV + 157 ILTETQQGL +++ 159 LLPLAPAAA ++ 161 SLIGIAIAL + 162 SLLDFLTFA + 163 SLMIDLIEV ++ 164 SLNPQEDVEF + 165 SLVDRVAAA +++ 166 VLFPLNLQL + 167 VLLDVALGL + 168 ALL + 169 VLQDPIWLL +++ 170 IVTEVAVGV +++ 171 DFL +++ 172 KLLWGDIMEL +++ 173 KMQETLVGL +++ 174 QW +++ 175 KMDJFLDMQL +++ 176 HLWTGEEQL +++ 177 KITTVIQHV +++ 178 KLWPLFVKL +++ 179 ENL +++ 180 ALDQEIIEV +++ 181 KLLNHVTQL +++ 183 SVIGVSPAV +++ 184 RMTDQEAIQDL +++ 185 RLIPIIVLL +++ 186 IILDEAHNV ++ 187 MLPPPPLTA ++ 188 RLLDFPTLL +++ EXAMPLE 2 Expression profiling of genes encoding the peptides of the invention resentation or specific presentation of a peptide on tumor cells compared to normal cells is sufficient for its usefulness in therapy, and some peptides are tumor-specific despite their source protein occurring also in normal tissues. Still, mRNA sion profiling adds an additional level of safety in selection of peptide s for immunotherapies. Especially for eutic options with high safety risks, such as affinity-matured TCRs, the ideal target peptide will be derived from a protein that is unique to the tumor and not found on normal tissues.

RNA sources and preparation Surgically removed tissue specimens were provided as indicated above (see e 1) after n informed consent had been obtained from each patient. Tumor tissue specimens were snap-frozen immediately after y and later homogenized with mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI Reagent (Ambion, adt, Germany) followed by a cleanup with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, MD, USA); Capital BioScience lnc. (Rockville, MD, USA); Geneticist lnc. (Glendale, CA, USA); lstituto Nazionale Tumori "Pascale" (Naples, Italy); ProteoGenex Inc. (Culver City, CA, USA); University Hospital Heidelberg (Heidelberg, Germany) Total RNA from tumor tissues for RNASeq experiments was ed from: Asterand (Detroit, MI, USA & Royston, Herts, UK); ProteoGenex Inc. (Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn, Germany) Quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit nt).

RNAseg ments Gene expression analysis of - tumor and normal tissue RNA samples was performed by next generation sequencing (RNAseq) by CeGaT (Ttibingen, Germany). Briefly, sequencing libraries are prepared using the na HiSeq v4 reagent kit according to the provider’s protocol (lllumina Inc, San Diego, CA, USA), which es RNA fragmentation, cDNA conversion and on of sequencing adaptors. Libraries derived from multiple samples are mixed lar and sequenced on the Illumina HiSeq 2500 sequencer ing to the manufacturer’s instructions, generating 50 bp single end reads. Processed reads are mapped to the human genome (GRCh38) using the STAR software. Expression data are provided on transcript level as RPKM (Reads Per Kilobase per Million mapped reads, generated by the software Cufflinks) and on exon level (total reads, generated by the software Bedtools), based on annotations of the ensembl sequence database (Ensembl77). Exon reads are normalized for exon length and alignment size to obtain RPKM values. Exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in AML are shown in Figure 2. sion scores for further exemplary genes are shown in Table 9.

Table 9: Expression . The table lists peptides from genes that are very highly over-expressed in tumors compared to a panel of normal tissues (+++), highly over- expressed in tumors compared to a panel of normal tissues (++) or over-expressed in tumors compared to a panel of normal tissues (+). The baseline for this score was calculated from measurements of the following relevant normal tissues: adipose , adrenal gland, artery, blood cells, bone marrow, brain, age, colon, esophagus, eye, gallbladder, heart, kidney, liver, lung, lymph node, pancreas, peripheral nerve, pituitary gland, rectum, ry gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, urinary bladder, vein. In case expression data for several samples of the same tissue type were available, the arithmetic mean of all respective samples was used for the calculation.

SEQ ID No Sequence Expression 1 LLDSAVYYL +++ 3 ALYDKTKRIFL +++ 4 FLPDAFVTM +++ FLYYEDLVSC +++ 8 ALLV + 12 ALIESNTAL +++ 16 FLDTQAPSL +++ 17 FLLGLSEQL + 36 VLGPSPSSV ++ 41 ALFILPFVSV ++ 42 ALTTYTIEV + 50 IIWATSLLL ++ 51 SLLSSSLNV ++ 60 NLLNVLEYL ++ 61 LLSEA ++ 63 QLL ++ 64 SLANIKIWV +++ 92 RLWDIQHQL + 96 TLLASYVFL +++ 97 TIMTGVIGV +++ 102 ERV +++ 103 ALGDFGIRL +++ 114 RLAQEAAQV + 146 ALDTRVVEL +++ 165 SLVDRVAAA ++ 175 KMDJFLDMQL ++ 176 HLWTGEEQL ++ 182 SLSEYDQCL +++ EXAMPLE 3 In vitro immunogenicity for MHC class I presented peptides In order to obtain information regarding the immunogenicity of the TUMAPs of the present invention, the inventors performed investigations using an in vitro T-cell priming assay based on repeated stimulations of CD8+ T cells with artificial antigen presenting cells (aAPCs) loaded with peptide/MHC complexes and anti-CD28 antibody. This way the inventors could show immunogenicity for 0201 restricted TUMAPs of the ion, demonstrating that these peptides are T-cell epitopes against which CD8+ precursor T cells exist in humans (Table 10).

In vitro priming of CD8+ T cells In order to perform in vitro stimulations by artificial antigen presenting cells loaded with peptide-MHC complex (pMHC) and D28 dy, the inventors first isolated CD8+ T cells from fresh HLA-A*02 leukapheresis products via positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of y donors obtained from the sity clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium (TCM) until use consisting of lutamax (lnvitrogen, Karlsruhe, Germany) supplemented with % heat inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 ug/ml Streptomycin ex, Cologne, Germany), 1 mM sodium te (CC Pro, Oberdorla, y), 20 pg/ml Gentamycin (Cambrex). 2.5 ng/ml IL- 7 (PromoCell, Heidelberg, Germany) and 10 U/ml lL-2 (Novartis Pharma, Ntirnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations and readout was performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung et al., 1987) was chemically biotinylated using Sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer (Perbio, Bonn, Germany). Beads used were 5.6 pm diameter streptavidin coated polystyrene particles (Bangs Laboratories, is, USA). pMHC used for positive and negative control stimulations were A*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 199) from modified A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 200), respectively. 800.000 beads / 200 pl were coated in 96-well plates in the presence of 4 x 12.5 ng different -pMHC, washed and 600 ng biotin anti-CD28 were added subsequently in a volume of 200 pl. Stimulations were initiated in 96-well plates by co-incubating 1x106 CD8+ T cells with 2x105 washed coated beads in 200 pl TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3 days at 37°C. Half of the medium was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and ting was continued for 4 days at 37°C. This stimulation cycle was performed for a total of three times. For the pMHC multimer readout using eight different pMHC molecules per condition, a two- dimensional atorial coding approach was used as previously described (Andersen et al., 2012) with minor modifications encompassing coupling to 5 different fluorochromes. Finally, multimeric analyses were performed by staining the cells with Live/dead near IR dye (lnvitrogen, Karlsruhe, Germany), CD8-FITC antibody clone 8K1 (BD, Heidelberg, Germany) and fluorescent pMHC multimers. For is, a BD LSRII SORP cytometer equipped with appropriate lasers and filters was used. Peptide specific cells were ated as percentage of total CD8+ cells. Evaluation of multimeric is was done using the FlowJo software (Tree Star, Oregon, USA). In vitro priming of specific multimer+ CD8+ cytes was detected by comparing to negative control stimulations. Immunogenicity for a given antigen was detected if at least one evaluable in vitro stimulated well of one y donor was found to contain a specific CD8+ T-cell line after in vitro stimulation (i.e. this well contained at least 1% of specific er+ among CD8+ T-cells and the percentage of specific multimer+ cells was at least 10x the median of the negative control stimulations).

In vitro immunogenicity for AML peptides For tested HLA class I peptides, in vitro immunogenicity could be demonstrated by generation of peptide specific T-cell lines. Exemplary flow cytometry results after TUMAP-specific multimer staining for 2 peptides of the invention are shown in Figure 3 together with corresponding negative controls. Results for 10 peptides from the invention are summarized in Table 10.

Table 10: in vitro immunogenicity of HLA class I peptides of the invention ary results of in vitro genicity experiments conducted by the applicant for the peptides of the invention. <20 % = +; 20 % - 49 % = ++; 50 % - 69 %= +++; >= 70 % = ++++ Seq ID NO: Sequence wells 189 RLFEEVLGV ++ 190 LSV ++ 191 ALSVLRLAL ++ 192 GLAALAVHL ++ 193 FLLAEDTKV ++ 194 PEI ++ 195 FLFVDPELV +++ 196 lLVDWLVQV ++ 197 VLLNEILEQV ++ 198 KIQEILTQV ++ EXAMPLE 4 Synthesis of peptides All es were synthesized using standard and well-established solid phase peptide synthesis using the Fmoc-strategy. Identity and purity of each dual peptide have been determined by mass spectrometry and analytical RP-HPLC. The peptides were obtained as white to off-white lyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs are preferably administered as trifluoro-acetate salts or acetate salts, other salt-forms are also possible.

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Claims (24)

1. A peptide comprising an amino acid ce selected from the group consisting of SEQ ID No. 4, SEQ ID No. 3, and SEQ ID No. 5, or a pharmaceutical acceptable salt thereof, wherein said e has an overall length of up to 16 amino acids.

2. The peptide ing to claim 1, wherein said peptide has the ability to bind to an MHC class-I or –II molecule, and wherein said peptide, when bound to said MHC, is capable of being recognized by CD4 and/or CD8 T cells.

3. The peptide according to claim 1 or claim 2, wherein said peptide consists of an amino acid sequence according to any one of SEQ ID No. 4, SEQ ID No. 3, and SEQ ID No. 5.

4. The e according to any one of claims 1 to 3, wherein said peptide includes non-peptide bonds.

5. The e according to any one of claims 1 to 4, wherein said peptide is part of a fusion protein.

6. The peptide according to claim 5, wherein said fusion protein comprises N-terminal amino acids of the HLA-DR antigen-associated invariant chain (Ii).

7. An antibody or fragment thereof that specifically recognizes the peptide according to any one of claims 1 to 4, or the peptide according to any of claims 1 to 4 when bound to an MHC molecule.

8. The antibody according to claim 7, wherein said antibody is a soluble or membranebound

9. The antibody according to claim 7 or claim 8, wherein said antibody is a monoclonal antibody.

10. A T-cell receptor or a fragment thereof, that is reactive with an HLA ligand, wherein said ligand is the peptide ing to any one of claims 1 to 4, or the peptide according to any of claims 1 to 4 when bound to an MHC molecule.

11. The T-cell receptor according to claim 10, n said T-cell receptor is soluble or membrane-bound.

12. The T -cell receptor according to claim 11, wherein said T-cell receptor is soluble and carries a further effector function, including an immune stimulating domain or toxin.

13. An aptamer that specifically recognizes the peptide ing to any one of claims 1 to 4, or specifically recognizes the peptide according to any of claims 1 to 4 when bound to an MHC molecule.

14. A nucleic acid encoding for the e according to any one of claims 1 to 4, the antibody or fragment thereof according to any one of claims 7 to 9, or the T-cell receptor or fragment thereof according to any one of claims 10 to 12.

15. The nucleic acid according to claim 14, wherein the nucleic acid is linked to a heterologous promoter sequence.

16. An expression vector which expresses the nucleic acid of claim 14 or claim 15.

17. A recombinant host cell comprising the peptide ing to any one of claims 1 to 4, the antibody or fragment thereof according to any one of claims 7 to 9, the T-cell receptor or fragment thereof according to any one of claims 10 to 12, the c acid according to claim 14 or claim 15, or the expression vector according to claim 16, wherein the host cell is not a cell within a human body.

18. The host cell according to claim 17, wherein said host cell is selected from an antigen ting cell, a dendritic cell, a T cell or an NK cell.

19. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro s with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate said T- cells in an n specific manner, wherein said antigen is a peptide according to any one of claims 1 to 3.

20. An activated T lymphocyte produced by the method according to claim 19, that specifically recognizes a cell which presents a polypeptide comprising an amino acid sequence given in any one of claims 1 to 3.

21. A ceutical ition comprising at least one active ingredient selected from the group consisting of the peptide according to any one of claims 1 to 5, the antibody or fragment thereof according to any one of claims 7 to 9, the T-cell receptor or fragment thereof according to any one of claims 10 to 12, the aptamer according to claim 13, the nucleic acid according to claim 14 or claim 15, the expression vector according to claim 16, the host cell ing to claim 17 or claim 18, the activated T lymphocyte ing to claim 20, or a conjugated or labelled active ingredient, and a pharmaceutically able carrier, and/or additional ceutically acceptable excipients and/or stabilizers.

22. A method for producing the peptide according to any one of claims 1 to 5, the antibody or fragment thereof according to any one of claims 7 to 9, or the T-cell receptor or fragment f according to any one of claims 10 to 12, the method comprising culturing the host cell ing to claim 17 or claim 18, and isolating the peptide, the antibody or fragment thereof, or the T cell or or fragment thereof, from said host cell and/or its culture medium.

23. Use of the e according to any one of claims 1 to 5, the antibody or fragment thereof according to any one of claims 7 to 9, the T-cell receptor or fragment thereof according to any one of claims 10 to 12, the aptamer according to claim 13, the nucleic acid according to claim 14 or claim 15, the expression vector ing to claim 16, the host cell according to claim 17 or claim 18, or the activated T lymphocyte according to claim 20, in the manufacture of a medicament against cancer.

24. The use according to claim 23, wherein said cancer is selected from the group of AML, bile duct cancer, brain cancer, breast cancer, chronic lymphocytic leukemia, colon or rectal cancer, esophageal cancer, gallbladder cancer, liver cancer, melanoma, non-hodgkin lymphoma, non-small cell lung , ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, small cell lung cancer, urinary bladder cancer, uterine cancer and other tumors that show an overexpression of a protein from which a peptide of SEQ ID No. 4, SEQ ID No. 3, or SEQ ID No. 5 is derived from.